Methods for aiding in the determination of whether to perform imaging on a human subject who has sustained or may have sustained an injury to the head using early biomarkers

ABSTRACT

Disclosed herein are methods that aid in the determination of whether to perform imaging, such as magnetic resonance imaging (MRI) or computerized tomography (CT) scan, on a human subject that has sustained or may have sustained an injury to the head using an early biomarker, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof. These methods involve detecting levels and changes in levels of UCH-L1 in samples taken from a human subject at time points within 24 hours after the subject has sustained or may have sustained an injury to the head.

RELATED APPLICATION INFORMATION

This application claims priority to U.S. Application No. 62/511,126filed on May 25, 2017, the contents of which are herein incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 17, 2018, isnamed 36101US2ORDST25.txt and is 6,612 bytes in size.

TECHNICAL FIELD

The present invention relates to methods of aiding in the determinationof whether to perform imaging, such as magnetic resonance imaging (MRI)or head computerized tomography (CT) scan, on a human subject that hassustained or may have sustained an injury to the head by detectingchanges in levels of an early biomarker, such as ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof, in samples taken from a human subjectat time points within 24 hours after the subject has sustained or mayhave sustained an injury to the head.

BACKGROUND

More than 5 million mild traumatic brain injuries (TBIs) occur each yearin the United States alone. Currently, there is no simple, objective,accurate measurement available to help in patient assessment. In fact,much of TBI evaluation and diagnosis is based on subjective data.Unfortunately, objective measurements such as head CT and Glasgow ComaScore (GCS) are not very comprehensive or sensitive in evaluating mildTBI. Moreover, head CT is unrevealing for the vast majority of the timefor mild TBI, is expensive, and exposes the patient to unnecessaryradiation. Additionally, a negative head CT does not mean the patienthas been cleared from having a concussion; rather it just means certaininterventions, such as surgery, are not warranted. Clinicians andpatients need objective, reliable information to accurately evaluatethis condition to promote appropriate triage and recovery. To date,limited data have been available for the use of UCH-L1 and GFAP in theacute care setting to aid in patient evaluation and management.

Mild TBI or concussion is much harder to objectively detect and presentsan everyday challenge in emergency care units globally. Concussionusually causes no gross pathology, such as hemorrhage, and noabnormalities on conventional computed tomography scans of the brain,but rather rapid-onset neuronal dysfunction that resolves in aspontaneous manner over a few days to a few weeks. Approximately 15% ofmild TBI patients suffer persisting cognitive dysfunction. There is anunmet need for mild TBI victims on scene, in emergency rooms andclinics, in the sports area and in military activity (e.g., combat).

Current algorithms for assessment of the severity of brain injuryinclude Glasgow Coma Scale score and other measures. These measures mayat times be adequate for relating acute severity but are insufficientlysensitive for subtle pathology which can result in persistent deficit.GCS and other measures also do not enable differentiation among types ofinjury and may not be adequate. Thus patients grouped into a single GCSlevel entering a clinical trial may have vastly heterogeneous severityand type of injury. Because outcomes also vary accordingly,inappropriate classification undermines the integrity of a clinicaltrial. Improved classification of injury will enable more precisedelineation of disease severity and type for TBI patients in clinicaltrials.

Additionally, current brain injury trials rely on outcome measures suchas Glasgow Outcome Scale Extended, which capture global phenomena butfail to assess for subtle differences in outcome. Thus 30 consecutivetrials for brain injury therapeutics have failed. Sensitive outcomemeasures are needed to determine how well patients have recovered frombrain injury in order to test therapeutics and prophylactics.

SUMMARY

In one aspect, the present disclosure is directed to a method of aidingin the determination of whether to perform an imaging procedure on ahuman subject that has sustained or may have sustained an injury to thehead. The method comprises: performing an assay on a sample obtainedfrom the subject within about 24 hours after a suspected injury to thehead to measure or detect a level of an early biomarker in the sample,said early biomarker comprising ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof, in the sample; and performing an imaging procedure on thesubject when the level of the early biomarker in the sample is higherthan a reference level of the early biomarker (“rule in” performing animaging procedure) and not performing an imaging procedure on thesubject when the level of the early biomarker in the sample is lowerthan a reference level of the early biomarker (“rule out” performing animaging procedure). The reference level is (a) determined by an assayhaving a sensitivity of between at least about 70% to 100% and aspecificity of between at least about 30% to 100%; or (b) between atleast about 20 pg/mL to about 200 pg/mL.

In another aspect, the present disclosure is directed to a method ofaiding in the determination of whether to perform an imaging procedureon a human subject that has sustained or may have sustained an injury tothe head. The method comprises: performing an assay on at least twosamples obtained from the subject, the first sample taken from thesubject within 24 hours of a suspected injury and the second sampletaken from the subject from about 3 to about 6 hours after the firstsample is taken; detecting in the at least two samples an earlybiomarker of traumatic brain injury, said early biomarker comprisingubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillaryacidic protein (GFAP), or a combination thereof; and performing animaging procedure on the subject when the level of the early biomarkerdecreases or increases by at least an absolute amount from the firstsample to the second sample (“rule in” performing an imaging procedure)and not performing an imaging procedure on the subject when there is nodecrease or increase by at least an absolute amount in the level of theearly biomarker from the first sample to the second sample (“rule out”performing an imaging procedure).

In still a further aspect, the present disclosure relates to a method ofaiding in the determination of whether to perform an imaging procedureon a human subject that has sustained or may have sustained an injury tothe head. The method comprises:

-   -   a) performing an assay on a sample obtained from the subject        within about 24 hours after a suspected injury to the head to        measure or detect a level of an early biomarker in the sample,        said early biomarker comprising ubiquitin carboxy-terminal        hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP),        or a combination thereof, in the sample; and    -   b) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker in the sample is        higher than a reference level of the early biomarker and not        performing an imaging procedure on the subject when the level of        the early biomarker in the sample is lower than a reference        level of the early biomarker,        wherein the reference level is (a) determined by an assay having        a sensitivity of between at least about 70% to 100% and a        specificity of between at least about 30% to 100%; or (b)        between at least about 20 pg/mL to about 200 pg/mL. In the event        that the early biomarker level is equal to the reference level,        then the result would be considered to negative, meaning that no        imaging would be performed.

In the above-described method, the reference level is determined by anassay having a sensitivity of at least about 80% and a specificity of atleast about 30%. In some embodiments, the reference level is between atleast about 80 pg/mL to about 150 pg/mL. More specifically, in someembodiments, the reference level for GFAP is between about 20 pg/mL andabout 200 pg/mL. In other embodiments, the reference level for UCH-L1 isabout 80 pg/mL and about 150 pg/mL.

In the above-described method for determining or evaluating whether toperform imaging, the subject may be suspected of having a traumaticbrain injury based on an imaging procedure, such as, for example, a MRIor CT scan, that has been or already was performed (meaning, prior tothe assay being performed). For example, depending upon a subject'smedical condition (such as, if the patient is unconscious), an imagingprocedure (such as an MRI or CT scan) may be conducted shortly after thesubject arrives at an emergency room, trauma center, or other site inorder to assess and/or evaluate whether the subject has a TBI. Such animaging procedure (such as an MRI or CT scan) may be performed prior tothe assay being performed to confirm and determine whether or not thesubject has a mild, moderate, severe, or moderate to severe TBI. Afterthe assay is performed, one or more subsequent imaging procedures (suchas one or more MRIs or CT scans)) can be performed based on the resultsof the assay as part of the physician's (or other medical personnel's)management of the TBI (such as, for example, to determine whethersurgical and/or pharmacological intervention may be required).

In certain embodiments of the above method, the subject may be suspectedof having a traumatic brain injury based on an imaging procedure (suchas a MRI or CT scan). For example, a subject may be suspected of havinga mild TBI based on an imaging procedure (such as a MRI or CT scan).Alternatively, a subject may be suspected of having a moderate TBI basedon an imaging procedure (such as a MRI or CT scan). Alternatively, asubject may be suspected of having a severe TBI based on an imagingprocedure (such as a MRI or CT scan). Alternatively, a subject may besuspected of having a moderate to severe TBI based on an imagingprocedure (such as a MRI or CT scan). Still further, a subject may besuspected of not having a TBI based on a MRI.

In certain embodiments of the above method, the reference level used iscorrelated or corresponds to a positive MRI. In other embodiments of theabove methods, the reference level is correlated with or corresponds toa positive head CT scan. In still further embodiments, the referencelevel is correlated with or corresponds to an intercranial lesion. Forexample, the reference level can correlate or correspond (such asthrough an increase or decrease in the reference level) to subjectshaving a positive MRI. Alternatively, the reference levels can correlateor correspond (such as through an increase or decrease in the referencelevel) to subjects having a positive head CT scan. Still further, thereference levels can correlate or correspond (such as through anincrease or decrease in the reference level) to subjects having anintercranial lesion. In other embodiments of the above method, thereference level is correlated or corresponds to control subjects whichhave not suffered any TBI.

In the above-described method, the sample can be taken within about 0 toabout 12 hours after a suspected injury to the head. For example, thesample can be taken within about 5 minutes after a suspected injury tothe head. Alternatively, the sample can be taken within about 10 minutesof a suspected injury to the head. Alternatively, the sample can betaken within about 12 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 15 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within about20 minutes of a suspected injury to the head. Alternatively, the samplecan be taken within 30 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 60 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within 1.5hours of a suspected injury to the head. Alternatively, the sample canbe taken within 2 hours of a suspected injury to the head.Alternatively, the sample can be taken within 3 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 4hours of a suspected injury to the head. Alternatively, the sample canbe taken within 5 hours of a suspected injury to the head.Alternatively, the sample can be taken within 6 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 7hours of a suspected injury to the head. Alternatively, the sample canbe taken within 8 hours of a suspected injury to the head.Alternatively, the sample can be taken within 9 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 10hours of a suspected injury to the head. Alternatively, the sample canbe taken within 11 hours of a suspected injury to the head.Alternatively, the sample can be taken within 12 hours of a suspectedinjury to the head.

In one embodiment, using the above-described method, the subject isassessed or evaluated as having a mild TBI. In another embodiment, usingthe above-described method, the subject is assessed or evaluated ashaving a moderate TBI. In another embodiment, using the above-describedmethod, the subject is assessed or evaluated as having a severe TBI. Inanother embodiment, using the above-described method, the subject isassessed or evaluated as having a moderate to severe TBI. In yet still afurther embodiment, using the above-described method, the subject isassessed or evaluated as not having a TBI.

The above-described method can further comprise treating a human subjectassessed or evaluated as having a mild, moderate, severe, or a moderateto severe TBI with a treatment for TBI (e.g., a surgical treatment, atherapeutic treatment, or combinations thereof). Any such treatmentknown in the art and described further herein can be used. Moreover, ina further embodiment, any subject being treated for TBI can also,optionally, be monitored during and after any course of treatment.Alternatively, said methods can further comprise monitoring a subjectassessed as having a mild, moderate, severe, or a moderate to severe TBI(such as those, who as of yet, may not be receiving any treatment).

In the above-described method, the sample can be selected from the groupconsisting of a whole blood sample, a serum sample, a cerebrospinalfluid sample, and a plasma sample. In some embodiments, the sample is awhole blood sample. In some embodiments, the sample is a plasma sample.In yet other embodiments, the sample is a serum sample. Such a samplecan be obtained in a variety of ways. For example, the sample can beobtained after the subject sustained a head injury caused by physicalshaking, blunt impact by an external mechanical or other force thatresults in a closed or open head trauma, one or more falls, explosionsor blasts or other types of blunt force trauma. Alternatively, thesample can be obtained after the subject has ingested or been exposed toa chemical, toxin or combination of a chemical and toxin. Examples ofchemicals or toxins are fire, mold, asbestos, a pesticide, aninsecticide, an organic solvent, a paint, a glue, a gas, an organicmetal, a drug of abuse or one or more combinations thereof. Stillfurther, the sample can be obtained from a subject that suffers from anautoimmune disease, a metabolic disorder, a brain tumor, hypoxia, avirus, meningitis, hydrocephalus or combinations thereof.

Any of the above-described method can be carried out on any humansubject without regard to factors selected from the group consisting ofthe human subject's clinical condition, the human subject's laboratoryvalues, the human subject's classification as suffering from mild,moderate, severe, or a moderate to severe TBI, the human subject'sexhibition of low or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP,and the timing of any event wherein the human subject may have sustainedhead injury.

In the above-described method, the assay is an immunoassay. In someembodiments, the assay is a point-of-care assay. In yet otherembodiments, the assay is a clinical chemistry assay. In yet otherembodiments, the assay is a single molecule detection assay. In yetother embodiments, the assay is an immunoassay, the subject is a humanand the sample is whole blood. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is wholeblood. In yet other embodiments, the assay is a clinical chemistry assayand the sample is whole blood. In still further embodiments, the assayis a single molecule detection assay and the sample is whole blood. Inyet other embodiments, the assay is an immunoassay, the subject is ahuman and the sample is serum. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is serum. Inyet other embodiments, the assay is a clinical chemistry assay and thesample is serum. In still further embodiments, the assay is a singlemolecule detection assay and the sample is serum. In yet otherembodiments, the assay is an immunoassay, the subject is a human and thesample is plasma. In yet other embodiments, the assay is a point-of-careassay, the subject is a human and the sample is plasma. In yet otherembodiments, the assay is a clinical chemistry assay and the sample isplasma. In still further embodiments, the assay is a single moleculedetection assay and the sample is plasma.

In yet another aspect, the present invention relates to a method ofevaluating a human subject to determine whether to perform an imagingprocedure for a head injury. The method comprises the steps of:

-   -   a) performing an assay on a sample obtained from the subject        within about 24 hours after a suspected injury to the head to        measure a level of an early biomarker in the sample, said early        biomarker comprising ubiquitin carboxy-terminal hydrolase L1        (UCH-L1), glial fibrillary acidic protein (GFAP), or a        combination thereof, in the sample; and    -   b) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker in the sample is        higher than a reference level of the early biomarker and not        performing an imaging procedure on the subject when the level of        the early biomarker in the sample is lower than a reference        level of the early biomarker,        wherein the reference level is (a) determined by an assay having        a sensitivity of between at least about 70% to 100% and a        specificity of between at least about 30% to 100%; or (b)        between at least about 20 pg/mL to about 200 pg/mL. In the event        that the early biomarker level is equal to the reference level,        then the result would be considered to negative, meaning that no        imaging would be performed.

In the above-described method, the reference level is determined by anassay having a sensitivity of at least about 80% and a specificity of atleast about 30%. In some embodiments, the reference level is between atleast about 80 pg/mL to about 150 pg/mL. More specifically, in someembodiments, the reference level for GFAP is between about 20 pg/mL andabout 200 pg/mL. In other embodiments, the reference level for UCH-L1 isabout 80 pg/mL and about 150 pg/mL.

In the above-described method for determining or evaluating whether toperform imaging, the subject may be suspected of having a traumaticbrain injury based on an imaging procedure, such as, for example, a MRIor CT scan, that has been or already was performed (meaning, prior tothe assay being performed). For example, depending upon a subject'smedical condition (such as, if the patient is unconscious), an imagingprocedure (such as an MRI or CT scan) may be conducted shortly after thesubject arrives at an emergency room, trauma center, or other site inorder to assess and/or evaluate whether the subject has a TBI. Such animaging procedure (such as an MRI or CT scan) may be performed prior tothe assay being performed to confirm and determine whether or not thesubject has a mild, moderate, severe, or moderate to severe TBI. Afterthe assay is performed, one or more subsequent imaging procedures (suchas one or more MRIs or CT scans)) can be performed based on the resultsof the assay as part of the physician's (or other medical personnel's)management of the TBI (such as, for example, to determine whethersurgical and/or pharmacological intervention may be required).

In certain embodiments of the above method, the subject may be suspectedof having a traumatic brain injury based on an imaging procedure (suchas a MRI or CT scan). For example, a subject may be suspected of havinga mild TBI based on an imaging procedure (such as a MRI or CT scan).Alternatively, a subject may be suspected of having a moderate TBI basedon an imaging procedure (such as a MRI or CT scan). Alternatively, asubject may be suspected of having a severe TBI based on an imagingprocedure (such as a MRI or CT scan). Alternatively, a subject may besuspected of having a moderate to severe TBI based on an imagingprocedure (such as a MRI or CT scan). Still further, a subject may besuspected of not having a TBI based on a MRI.

In certain embodiments of the above method, the reference level used iscorrelated or corresponds to a positive MRI. In other embodiments of theabove methods, the reference level is correlated with or corresponds toa positive head CT scan. In still further embodiments, the referencelevel is correlated with or corresponds to an intercranial lesion. Forexample, the reference level can correlate or correspond (such asthrough an increase or decrease in the reference level) to subjectshaving a positive MRI. Alternatively, the reference levels can correlateor correspond (such as through an increase or decrease in the referencelevel) to subjects having a positive head CT scan. Still further, thereference levels can correlate or correspond (such as through anincrease or decrease in the reference level) to subjects having anintercranial lesion. In other embodiments of the above method, thereference level is correlated or corresponds to control subjects whichhave not suffered any TBI.

In the above-described method, the sample can be taken within about 0 toabout 12 hours after a suspected injury to the head. For example, thesample can be taken within about 5 minutes after a suspected injury tothe head. Alternatively, the sample can be taken within about 10 minutesof a suspected injury to the head. Alternatively, the sample can betaken within about 12 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 15 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within about20 minutes of a suspected injury to the head. Alternatively, the samplecan be taken within 30 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 60 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within 1.5hours of a suspected injury to the head. Alternatively, the sample canbe taken within 2 hours of a suspected injury to the head.Alternatively, the sample can be taken within 3 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 4hours of a suspected injury to the head. Alternatively, the sample canbe taken within 5 hours of a suspected injury to the head.Alternatively, the sample can be taken within 6 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 7hours of a suspected injury to the head. Alternatively, the sample canbe taken within 8 hours of a suspected injury to the head.Alternatively, the sample can be taken within 9 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 10hours of a suspected injury to the head. Alternatively, the sample canbe taken within 11 hours of a suspected injury to the head.Alternatively, the sample can be taken within 12 hours of a suspectedinjury to the head.

In one embodiment, using the above-described method, the subject isassessed or evaluated as having a mild TBI. In another embodiment, usingthe above-described methods, the subject is assessed or evaluated ashaving a moderate TBI. In another embodiment, using the above-describedmethod, the subject is assessed or evaluated as having a severe TBI. Inanother embodiment, using the above-described method, the subject isassessed or evaluated as having a moderate to severe TBI. In yet still afurther embodiment, using the above-described method, the subject isassessed or evaluated as not having a TBI.

The above-described method can further comprise treating a human subjectassessed or evaluated as having a mild, moderate, severe, or a moderateto severe TBI with a treatment for TBI (e.g., a surgical treatment, atherapeutic treatment, or combinations thereof). Any such treatmentknown in the art and described further herein can be used. Moreover, ina further embodiment, any subject being treated for TBI can also,optionally, be monitored during and after any course of treatment.Alternatively, said method can further comprise monitoring a subjectassessed as having a mild, moderate, severe, or a moderate to severe TBI(such as those, who as of yet, may not be receiving any treatment).

In the above-described method, the sample can be selected from the groupconsisting of a whole blood sample, a serum sample, a cerebrospinalfluid sample, and a plasma sample. In some embodiments, the sample is awhole blood sample. In some embodiments, the sample is a plasma sample.In yet other embodiments, the sample is a serum sample. Such a samplecan be obtained in a variety of ways. For example, the sample can beobtained after the subject sustained a head injury caused by physicalshaking, blunt impact by an external mechanical or other force thatresults in a closed or open head trauma, one or more falls, explosionsor blasts or other types of blunt force trauma. Alternatively, thesample can be obtained after the subject has ingested or been exposed toa chemical, toxin or combination of a chemical and toxin. Examples ofchemicals or toxins are fire, mold, asbestos, a pesticide, aninsecticide, an organic solvent, a paint, a glue, a gas, an organicmetal, a drug of abuse or one or more combinations thereof. Stillfurther, the sample can be obtained from a subject that suffers from anautoimmune disease, a metabolic disorder, a brain tumor, hypoxia, avirus, meningitis, hydrocephalus or combinations thereof.

The above-described method can be carried out on any human subjectwithout regard to factors selected from the group consisting of thehuman subject's clinical condition, the human subject's laboratoryvalues, the human subject's classification as suffering from mild,moderate, severe, or a moderate to severe TBI, the human subject'sexhibition of low or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP,and the timing of any event wherein the human subject may have sustainedhead injury.

In the above-described method, the assay is an immunoassay. In someembodiments, the assay is a point-of-care assay. In yet otherembodiments, the assay is a clinical chemistry assay. In yet otherembodiments, the assay is a single molecule detection assay. In yetother embodiments, the assay is an immunoassay, the subject is a humanand the sample is whole blood. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is wholeblood. In yet other embodiments, the assay is a clinical chemistry assayand the sample is whole blood. In still further embodiments, the assayis a single molecule detection assay and the sample is whole blood. Inyet other embodiments, the assay is an immunoassay, the subject is ahuman and the sample is serum. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is serum. Inyet other embodiments, the assay is a clinical chemistry assay and thesample is serum. In still further embodiments, the assay is a singlemolecule detection assay and the sample is serum. In yet otherembodiments, the assay is an immunoassay, the subject is a human and thesample is plasma. In yet other embodiments, the assay is a point-of-careassay, the subject is a human and the sample is plasma. In yet otherembodiments, the assay is a clinical chemistry assay and the sample isplasma. In still further embodiments, the assay is a single moleculedetection assay and the sample is plasma.

In still a further aspect, the present disclosure relates to a method ofaiding in the determination of whether to perform an imaging procedureon a human subject that has sustained or may have sustained an injury tothe head. The method comprises:

-   -   a) performing an assay on at least two samples obtained from the        subject, the first sample taken from the subject within 24 hours        of a suspected injury and the second sample taken from the        subject from about 3 to about 6 hours after the first sample is        taken;    -   b) detecting in the at least two samples an early biomarker of        traumatic brain injury, said early biomarker comprising        ubiquitin carboxy-terminal hydrolase L1 (UCH-L 1), glial        fibrillary acidic protein (GFAP), or a combination thereof; and    -   c) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker decreases or        increases by at least an absolute amount from the first sample        to the second sample and not performing an imaging procedure on        the subject when there is no decrease or increase by at least an        absolute amount in the level of the early biomarker from the        first sample to the second sample.

In the above-described method, the absolute amount is determined by anassay having a sensitivity of between at least about 70% and aspecificity of at least about 100% and a sensitivity of between at leastabout 30% to 100%. In some embodiments, the absolute amount is betweenat least about 10 pg/mL to about 150 pg/mL. More specifically, in someembodiments, the absolute amount for GFAP is between about 30 pg/mL andabout 100 pg/mL. In other embodiments, the absolute amount for UCH-L1 isabout 10 pg/mL and about 150 pg/mL.

In the above described method, the measuring the level of UCH-L1comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a UCH-L1-capture antibody, which binds to an epitope on            UCH-L1 or UCH-L1 fragment to form a UCH-L1-capture            antibody-UCH-L1 antigen complex, and        -   (2) a UCH-L1-detection antibody which includes a detectable            label and binds to an epitope on UCH-L1 that is not bound by            the UCH-L1-capture antibody, to form a UCH-L1            antigen-UCH-L1-detection antibody complex,        -   such that a UCH-L1-capture antibody-UCH-L1            antigen-UCH-L1-detection antibody complex is formed, and    -   B. measuring the amount or concentration of UCH-L1 in the sample        based on the signal generated by the detectable label in the        UCH-L1-capture antibody-UCH-L1 antigen-UCH-L1-detection antibody        complex.

Alternatively, in the above-described method, the measuring of the levelof GFAP comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a GFAP-capture antibody, which binds to an epitope on            GFAP or GFAP fragment to form a GFAP-capture antibody-GFAP            antigen complex, and        -   (2) a GFAP-detection antibody which includes a detectable            label and binds to an epitope on GFAP that is not bound by            the GFAP-capture antibody, to form a GFAP            antigen-GFAP-detection antibody complex,        -   such that a GFAP-capture antibody-GFAP            antigen-GFAP-detection antibody complex is formed, and    -   B. measuring the amount or concentration of GFAP in the sample        based on the signal generated by the detectable label in the        GFAP-capture antibody-GFAP antigen-GFAP-detection antibody        complex.

In the above-described method for determining or evaluating whether toperform imaging, the subject may be suspected of having a traumaticbrain injury based on an imaging procedure, such as, for example, a MRIor CT scan, that has been or already was performed (meaning, prior tothe assay being performed). For example, depending upon a subject'smedical condition (such as, if the patient is unconscious), an imagingprocedure (such as an MRI or CT scan) may be conducted shortly after thesubject arrives at an emergency room, trauma center, or other site inorder to assess and/or evaluate whether the subject has a TBI. Such animaging procedure (such as an MRI or CT scan) may be performed prior tothe assay being performed to confirm and determine whether or not thesubject has a mild, moderate, severe, or moderate to severe TBI. Afterthe assay is performed, one or more subsequent imaging procedures (suchas one or more MRIs or CT scans)) can be performed based on the resultsof the assay as part of the physician's (or other medical personnel's)management of the TBI (such as, for example, to determine whethersurgical and/or pharmacological intervention may be required).

In certain embodiments of the above method, the subject may be suspectedof having a traumatic brain injury based on an imaging procedure (suchas a MRI or CT scan). For example, a subject may be suspected of havinga mild TBI based on an imaging procedure (such as a MRI or CT scan).Alternatively, a subject may be suspected of having a moderate TBI basedon an imaging procedure (such as a MRI or CT scan). Alternatively, asubject may be suspected of having a severe TBI based on an imagingprocedure (such as a MRI or CT scan). Alternatively, a subject may besuspected of having a moderate to severe TBI based on an imagingprocedure (such as a MRI or CT scan). Still further, a subject may besuspected of not having a TBI based on a MRI.

In certain embodiments of the above method, the reference level used iscorrelated or corresponds to a positive MRI. In other embodiments of theabove method, the reference level is correlated with or corresponds to apositive head CT scan. In still further embodiments, the reference levelis correlated with or corresponds to an intercranial lesion. Forexample, the reference level can correlate or correspond (such asthrough an increase or decrease in the reference level) to subjectshaving a positive MRI. Alternatively, the reference levels can correlateor correspond (such as through an increase or decrease in the referencelevel) to subjects having a positive head CT scan. Still further, thereference levels can correlate or correspond (such as through anincrease or decrease in the reference level) to subjects having anintercranial lesion. In other embodiments of the above method, thereference level is correlated or corresponds to control subjects whichhave not suffered any TBI.

In the above-described methods, the sample can be taken within about 0to about 12 hours after a suspected injury to the head. For example, thesample can be taken within about 5 minutes after a suspected injury tothe head. Alternatively, the sample can be taken within about 10 minutesof a suspected injury to the head. Alternatively, the sample can betaken within about 12 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 15 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within about20 minutes of a suspected injury to the head. Alternatively, the samplecan be taken within 30 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 60 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within 1.5hours of a suspected injury to the head. Alternatively, the sample canbe taken within 2 hours of a suspected injury to the head.Alternatively, the sample can be taken within 3 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 4hours of a suspected injury to the head. Alternatively, the sample canbe taken within 5 hours of a suspected injury to the head.Alternatively, the sample can be taken within 6 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 7hours of a suspected injury to the head. Alternatively, the sample canbe taken within 8 hours of a suspected injury to the head.Alternatively, the sample can be taken within 9 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 10hours of a suspected injury to the head. Alternatively, the sample canbe taken within 11 hours of a suspected injury to the head.Alternatively, the sample can be taken within 12 hours of a suspectedinjury to the head.

In one embodiment, using the above-described method, the subject isassessed or evaluated as having a mild TBI. In another embodiment, usingthe above-described method, the subject is assessed or evaluated ashaving a moderate TBI. In another embodiment, using the above-describedmethod, the subject is assessed or evaluated as having a severe TBI. Inanother embodiment, using the above-described method, the subject isassessed or evaluated as having a moderate to severe TBI. In yet still afurther embodiment, using the above-described method, the subject isassessed or evaluated as not having a TBI.

The above-described method can further comprise treating a human subjectassessed or evaluated as having a mild, moderate, severe, or a moderateto severe TBI with a treatment for TBI (e.g., a surgical treatment, atherapeutic treatment, or combinations thereof). Any such treatmentknown in the art and described further herein can be used. Moreover, ina further embodiment, any subject being treated for TBI can also,optionally, be monitored during and after any course of treatment.Alternatively, said method can further comprise monitoring a subjectassessed as having a mild, moderate, severe, or a moderate to severe TBI(such as those, who as of yet, may not be receiving any treatment).

In the above-described method, the sample can be selected from the groupconsisting of a whole blood sample, a serum sample, a cerebrospinalfluid sample, and a plasma sample. In some embodiments, the sample is awhole blood sample. In some embodiments, the sample is a plasma sample.In yet other embodiments, the sample is a serum sample. Such a samplecan be obtained in a variety of ways. For example, the sample can beobtained after the subject sustained a head injury caused by physicalshaking, blunt impact by an external mechanical or other force thatresults in a closed or open head trauma, one or more falls, explosionsor blasts or other types of blunt force trauma. Alternatively, thesample can be obtained after the subject has ingested or been exposed toa chemical, toxin or combination of a chemical and toxin. Examples ofchemicals or toxins are fire, mold, asbestos, a pesticide, aninsecticide, an organic solvent, a paint, a glue, a gas, an organicmetal, a drug of abuse or one or more combinations thereof. Stillfurther, the sample can be obtained from a subject that suffers from anautoimmune disease, a metabolic disorder, a brain tumor, hypoxia, avirus, meningitis, hydrocephalus or combinations thereof.

The above-described method can be carried out on any human subjectwithout regard to factors selected from the group consisting of thehuman subject's clinical condition, the human subject's laboratoryvalues, the human subject's classification as suffering from mild,moderate, severe, or a moderate to severe TBI, the human subject'sexhibition of low or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP,and the timing of any event wherein the human subject may have sustainedhead injury.

In the above-described method, the assay is an immunoassay. In someembodiments, the assay is a point-of-care assay. In yet otherembodiments, the assay is a clinical chemistry assay. In yet otherembodiments, the assay is a single molecule detection assay. In yetother embodiments, the assay is an immunoassay, the subject is a humanand the sample is whole blood. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is wholeblood. In yet other embodiments, the assay is a clinical chemistry assayand the sample is whole blood. In still further embodiments, the assayis a single molecule detection assay and the sample is whole blood. Inyet other embodiments, the assay is an immunoassay, the subject is ahuman and the sample is serum. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is serum. Inyet other embodiments, the assay is a clinical chemistry assay and thesample is serum. In still further embodiments, the assay is a singlemolecule detection assay and the sample is serum. In yet otherembodiments, the assay is an immunoassay, the subject is a human and thesample is plasma. In yet other embodiments, the assay is a point-of-careassay, the subject is a human and the sample is plasma. In yet otherembodiments, the assay is a clinical chemistry assay and the sample isplasma. In still further embodiments, the assay is a single moleculedetection assay and the sample is plasma.

In still a further aspect, the present disclosure relates to a method ofevaluating a human subject to determine whether to perform an imagingprocedure for a head injury. The method comprises the steps of:

-   -   a) performing an assay on at least two samples obtained from the        subject, the first sample taken from the subject within 24 hours        of a suspected injury and the second sample taken from the        subject from about 3 to about 6 hours after the first sample is        taken;    -   b) detecting in the at least two samples an early biomarker of        traumatic brain injury, said early biomarker comprising        ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial        fibrillary acidic protein (GFAP), or a combination thereof; and    -   c) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker decreases or        increases by at least an absolute amount from the first sample        to the second sample and not performing an imaging procedure on        the subject when there is no decrease or increase by at least an        absolute amount in the level of the early biomarker from the        first sample to the second sample.

In the above-described method, the absolute amount is determined by anassay having a sensitivity of between at least about 70% and aspecificity of at least about 100% and a sensitivity of between at leastabout 30% to 100%. In some embodiments, the absolute amount is betweenat least about 10 pg/mL to about 150 pg/mL. More specifically, in someembodiments, the absolute amount for GFAP is between about 30 pg/mL andabout 100 pg/mL. In other embodiments, the absolute amount for UCH-L1 isabout 10 pg/mL and about 150 pg/mL.

In the above described method, the measuring the level of UCH-L1comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a UCH-L1-capture antibody, which binds to an epitope on            UCH-L1 or UCH-L1 fragment to form a UCH-L1-capture            antibody-UCH-L1 antigen complex, and        -   (2) a UCH-L1-detection antibody which includes a detectable            label and binds to an epitope on UCH-L1 that is not bound by            the UCH-L1-capture antibody, to form a UCH-L1            antigen-UCH-L1-detection antibody complex,        -   such that a UCH-L1-capture antibody-UCH-L1            antigen-UCH-L1-detection antibody complex is formed, and    -   B. measuring the amount or concentration of UCH-L1 in the sample        based on the signal generated by the detectable label in the        UCH-L1-capture antibody-UCH-L1 antigen-UCH-L1-detection antibody        complex.

Alternatively, in the above-described method, the measuring of the levelof GFAP comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a GFAP-capture antibody, which binds to an epitope on            GFAP or GFAP fragment to form a GFAP-capture antibody-GFAP            antigen complex, and        -   (2) a GFAP-detection antibody which includes a detectable            label and binds to an epitope on GFAP that is not bound by            the GFAP-capture antibody, to form a GFAP            antigen-GFAP-detection antibody complex,        -   such that a GFAP-capture antibody-GFAP            antigen-GFAP-detection antibody complex is formed, and    -   B. measuring the amount or concentration of GFAP in the sample        based on the signal generated by the detectable label in the        GFAP-capture antibody-GFAP antigen-GFAP-detection antibody        complex.

In the above-described method for determining or evaluating whether toperform imaging, the subject may be suspected of having a traumaticbrain injury based on an imaging procedure, such as, for example, a MRIor CT scan, that has been or already was performed (meaning, prior tothe assay being performed). For example, depending upon a subject'smedical condition (such as, if the patient is unconscious), an imagingprocedure (such as an MRI or CT scan) may be conducted shortly after thesubject arrives at an emergency room, trauma center, or other site inorder to assess and/or evaluate whether the subject has a TBI. Such animaging procedure (such as an MRI or CT scan) may be performed prior tothe assay being performed to confirm and determine whether or not thesubject has a mild, moderate, severe, or moderate to severe TBI. Afterthe assay is performed, one or more subsequent imaging procedures (suchas one or more MRIs or CT scans)) can be performed based on the resultsof the assay as part of the physician's (or other medical personnel's)management of the TBI (such as, for example, to determine whethersurgical and/or pharmacological intervention may be required).

In certain embodiments of the above method, the subject may be suspectedof having a traumatic brain injury based on an imaging procedure (suchas a MRI or CT scan). For example, a subject may be suspected of havinga mild TBI based on an imaging procedure (such as a MRI or CT scan).Alternatively, a subject may be suspected of having a moderate TBI basedon an imaging procedure (such as a MRI or CT scan). Alternatively, asubject may be suspected of having a severe TBI based on an imagingprocedure (such as a MRI or CT scan). Alternatively, a subject may besuspected of having a moderate to severe TBI based on an imagingprocedure (such as a MRI or CT scan). Still further, a subject may besuspected of not having a TBI based on a MRI.

In certain embodiments of the above method, the reference level used iscorrelated or corresponds to a positive MRI. In other embodiments of theabove method, the reference level is correlated with or corresponds to apositive head CT scan. In still further embodiments, the reference levelis correlated with or corresponds to an intercranial lesion. Forexample, the reference level can correlate or correspond (such asthrough an increase or decrease in the reference level) to subjectshaving a positive MRI. Alternatively, the reference levels can correlateor correspond (such as through an increase or decrease in the referencelevel) to subjects having a positive head CT scan. Still further, thereference levels can correlate or correspond (such as through anincrease or decrease in the reference level) to subjects having anintercranial lesion. In other embodiments of the above method, thereference level is correlated or corresponds to control subjects whichhave not suffered any TBI.

In the above-described method, the sample can be taken within about 0 toabout 12 hours after a suspected injury to the head. For example, thesample can be taken within about 5 minutes after a suspected injury tothe head. Alternatively, the sample can be taken within about 10 minutesof a suspected injury to the head. Alternatively, the sample can betaken within about 12 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 15 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within about20 minutes of a suspected injury to the head. Alternatively, the samplecan be taken within 30 minutes of a suspected injury to the head.Alternatively, the sample can be taken within 60 minutes of a suspectedinjury to the head. Alternatively, the sample can be taken within 1.5hours of a suspected injury to the head. Alternatively, the sample canbe taken within 2 hours of a suspected injury to the head.Alternatively, the sample can be taken within 3 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 4hours of a suspected injury to the head. Alternatively, the sample canbe taken within 5 hours of a suspected injury to the head.Alternatively, the sample can be taken within 6 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 7hours of a suspected injury to the head. Alternatively, the sample canbe taken within 8 hours of a suspected injury to the head.Alternatively, the sample can be taken within 9 hours of a suspectedinjury to the head. Alternatively, the sample can be taken within 10hours of a suspected injury to the head. Alternatively, the sample canbe taken within 11 hours of a suspected injury to the head.Alternatively, the sample can be taken within 12 hours of a suspectedinjury to the head.

In one embodiment, using the above-described method, the subject isassessed or evaluated as having a mild TBI. In another embodiment, usingthe above-described method, the subject is assessed or evaluated ashaving a moderate TBI. In another embodiment, using the above-describedmethod, the subject is assessed or evaluated as having a severe TBI. Inanother embodiment, using the above-described method, the subject isassessed or evaluated as having a moderate to severe TBI. In yet still afurther embodiment, using the above-described method, the subject isassessed or evaluated as not having a TBI.

The above-described method can further comprise treating a human subjectassessed or evaluated as having a mild, moderate, severe, or a moderateto severe TBI with a treatment for TBI (e.g., a surgical treatment, atherapeutic treatment, or combinations thereof). Any such treatmentknown in the art and described further herein can be used. Moreover, ina further embodiment, any subject being treated for TBI can also,optionally, be monitored during and after any course of treatment.Alternatively, said method can further comprise monitoring a subjectassessed as having a mild, moderate, severe, or a moderate to severe TBI(such as those, who as of yet, may not be receiving any treatment).

In the above-described method, the sample can be selected from the groupconsisting of a whole blood sample, a serum sample, a cerebrospinalfluid sample, and a plasma sample. In some embodiments, the sample is awhole blood sample. In some embodiments, the sample is a plasma sample.In yet other embodiments, the sample is a serum sample. Such a samplecan be obtained in a variety of ways. For example, the sample can beobtained after the subject sustained a head injury caused by physicalshaking, blunt impact by an external mechanical or other force thatresults in a closed or open head trauma, one or more falls, explosionsor blasts or other types of blunt force trauma. Alternatively, thesample can be obtained after the subject has ingested or been exposed toa chemical, toxin or combination of a chemical and toxin. Examples ofchemicals or toxins are fire, mold, asbestos, a pesticide, aninsecticide, an organic solvent, a paint, a glue, a gas, an organicmetal, a drug of abuse or one or more combinations thereof. Stillfurther, the sample can be obtained from a subject that suffers from anautoimmune disease, a metabolic disorder, a brain tumor, hypoxia, avirus, meningitis, hydrocephalus or combinations thereof.

The above-described method can be carried out on any human subjectwithout regard to factors selected from the group consisting of thehuman subject's clinical condition, the human subject's laboratoryvalues, the human subject's classification as suffering from mild,moderate, severe, or a moderate to severe TBI, the human subject'sexhibition of low or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP,and the timing of any event wherein the human subject may have sustainedhead injury.

In the above-described method, the assay is an immunoassay. In someembodiments, the assay is a point-of-care assay. In yet otherembodiments, the assay is a clinical chemistry assay. In yet otherembodiments, the assay is a single molecule detection assay. In yetother embodiments, the assay is an immunoassay, the subject is a humanand the sample is whole blood. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is wholeblood. In yet other embodiments, the assay is a clinical chemistry assayand the sample is whole blood. In still further embodiments, the assayis a single molecule detection assay and the sample is whole blood. Inyet other embodiments, the assay is an immunoassay, the subject is ahuman and the sample is serum. In yet other embodiments, the assay is apoint-of-care assay, the subject is a human and the sample is serum. Inyet other embodiments, the assay is a clinical chemistry assay and thesample is serum. In still further embodiments, the assay is a singlemolecule detection assay and the sample is serum. In yet otherembodiments, the assay is an immunoassay, the subject is a human and thesample is plasma. In yet other embodiments, the assay is a point-of-careassay, the subject is a human and the sample is plasma. In yet otherembodiments, the assay is a clinical chemistry assay and the sample isplasma. In still further embodiments, the assay is a single moleculedetection assay and the sample is plasma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows biomarker UCH-L1 result s vs. time from injury from anexemplary subset of subjects.

FIG. 1B shows biomarker GFAP results vs. time from injury from anexemplary subset of subjects.

FIGS. 2A and 2B show receiver operating characteristic (ROC) analysis ofUCH-L1 levels (FIG. 2A) and GFAP levels (FIG. 2B) correlated with apresent or absent intracranial lesion result using MRI by time point.The sample is taken within 24 hours of head injury.

FIG. 3A shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of UCH-L1 levels (i.e., the absolutedifference between UCH-L1 levels at Time Point 2 and UCH-L1 levels atTime Point 1) correlated with a present or absent intracranial lesionresult using MRI. The sample at Time Point 1 is taken within 24 hours ofhead injury while the sample at Time Point 2 is taken about 3 to about 6hours after the Time Point 1 sample is taken.

FIG. 3B shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of GFAP levels (i.e., the absolutedifference between GFAP levels at Time Point 2 and GFAP levels at TimePoint 1) correlated with a present or absent intracranial lesion resultusing MRI. The sample at Time Point 1 is taken within 24 hours of headinjury while the sample at Time Point 2 is taken about 3 to about 6hours after the Time Point 1 sample is taken.

FIGS. 4A and 4B show receiver operating characteristic (ROC) analysis ofUCH-L1 levels (FIG. 4A) and GFAP levels (FIG. 4B) correlated with apresent or absent intracranial lesion result using CT scan by timepoint. The sample is taken within 24 hours of head injury.

FIG. 5A shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of UCH-L1 levels (i.e., the absolutedifference between UCH-L1 levels at Time Point 2 and UCH-L1 levels atTime Point 1) correlated with a present or absent intracranial lesionresult using CT scan. The sample at Time Point 1 is taken within 24hours of head injury while the sample at Time Point 2 is taken about 3to about 6 hours after the Time Point 1 sample is taken.

FIG. 5B shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of GFAP levels (i.e., the absolutedifference between GFAP levels at Time Point 2 and GFAP levels at TimePoint 1) correlated with a present or absent intracranial lesion resultusing CT scan. The sample at Time Point 1 is taken within 24 hours ofhead injury while the sample at Time Point 2 is taken about 3 to about 6hours after the Time Point 1 sample is taken.

FIGS. 6A and 6B show receiver operating characteristic (ROC) analysis ofUCH-L1 levels (FIG. 6A) and GFAP levels (FIG. 6B) correlated with apresent or absent intracranial lesion result using MRI or CT scan bytime point. The sample is taken within 24 hours of head injury.

FIG. 7A shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of UCH-L1 levels (i.e., the absolutedifference between UCH-L1 levels at Time Point 2 and UCH-L1 levels atTime Point 1) correlated with a present or absent intracranial lesionresult using MRI or CT scan. The sample at Time Point 1 is taken within24 hours of head injury while the sample at Time Point 2 is taken about3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 7B shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of GFAP levels (i.e., the absolutedifference between GFAP levels at Time Point 2 and GFAP levels at TimePoint 1) correlated with a present or absent intracranial lesion resultusing MRI or CT scan. The sample at Time Point 1 is taken within 24hours of head injury while the sample at Time Point 2 is taken about 3to about 6 hours after the Time Point 1 sample is taken.

FIGS. 8A and 8B show receiver operating characteristic (ROC) analysis ofUCH-L1 levels (FIG. 8A) and GFAP levels (FIG. 8B) correlated with apresent intracranial lesion result using MRI but negative CT scan bytime point. The sample is taken within 24 hours of head injury.

FIG. 9A shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of UCH-L1 levels (i.e., the absolutedifference between UCH-L1 levels at Time Point 2 and UCH-L1 levels atTime Point 1) correlated with a present intracranial lesion result usingMRI but negative CT scan. The sample at Time Point 1 is taken within 24hours of head injury while the sample at Time Point 2 is taken about 3to about 6 hours after the Time Point 1 sample is taken.

FIG. 9B shows a receiver operating characteristic (ROC) analysis ofabsolute amount (“absolute delta”) of GFAP levels (i.e., the absolutedifference between GFAP levels at Time Point 2 and GFAP levels at TimePoint 1) correlated with a present intracranial lesion result using MRIbut negative CT scan. The sample at Time Point 1 is taken within 24hours of head injury while the sample at Time Point 2 is taken about 3to about 6 hours after the Time Point 1 sample is taken.

DETAILED DESCRIPTION

The present disclosure relates to methods that aid in determiningwhether a human subject that has or may have sustained an injury to thehead would benefit from and thus receive an imaging procedure, such asmagnetic resonance imaging (MRI) or head computerized tomography (CT)scan, based on the levels of an early biomarker, such as UCH-L1, GFAP,or combination thereof. These methods involve detecting levels of theearly biomarker, such as UCH-L1, GFAP, or combination thereof, in one ormore samples taken from the human subject at a time point within about24 hours, e.g., 0 to about 12 hours, of the injury to the head orsuspected injury to the head. The detection levels of the earlybiomarker, such as UCH-L1, GFAP, or combination thereof, that are higherthan reference levels of the early biomarker, within about the first 24hours after injury or suspected injury to the head provides an aid inthe determination of whether a human subject should receive an imagingprocedure, i.e., “rule in” an imaging procedure. For example, humansubjects having a level of the early biomarker, such as UCH-L1, GFAP, orcombination thereof, higher than a reference level of the earlybiomarker, such as UCH-L1, GFAP, or a combination thereof, may also beidentified as likely to have a positive head CT scan or a positive MRI,i.e., a intracranial lesion present, (e.g., thus indicating a potentialTBI) and thus benefit from having a head CT scan or MRI. Alternatively,certain levels of the early biomarker can be used to “rule out” a needfor an imaging procedure. For example, human subjects having a level ofthe early biomarker, such as UCH-L1, GFAP, or combination thereof, lowerthan a reference level of the early biomarker, such as UCH-L1, GFAP, ora combination thereof, may be identified as likely to have a negativehead CT scan or a negative MRI, i.e., an absence of an intracraniallesion, and thus a head CT scan or MRI would not be needed or performed.

The present invention relates to methods that involve detecting levelsof the early biomarker, such as UCH-L1, GFAP, or combination thereof, inone or more samples taken from the human subject at different timepoints within 24 hours of the injury to the head or suspected injury tothe head. The detection of an increase in or elevated levels of theearly biomarker, such as UCH-L1, GFAP, or combination thereof, that maybe followed by a subsequent decrease in the levels of the earlybiomarker within about the first 24 hours after injury or suspectedinjury to the head provides an aid in determining whether a humansubject should receive an imaging procedure, such as MRI or head CTscan. For example, human subjects having at least an increase ordecrease of an early biomarker, such as UCH-L1, GFAP, or combinationthereof, by an absolute amount may also be identified as likely to havea positive head CT scan or a positive MRI, i.e., a intracranial lesionpresent, (e.g., thus indicating a potential TBI) and thus benefit fromhaving a head CT scan or MRI, i.e., “rule in” an imaging procedure.Alternatively, no decrease or increase by at least an absolute amount ofthe early biomarker can be used to “rule out” a need for an imagingprocedure. For example, human subjects having a change in levels of theearly biomarker, such as UCH-L1, GFAP, or combination thereof, less thanan absolute amount may be identified as likely to have a negative headCT scan or a negative MRI, i.e., an absence of an intracranial lesion,and thus a head CT scan or MRI would not be needed or performed.

For avoidance of any doubt, the one or more samples taken from the humansubject at a time point within about 24 hours refers to the fact thatthe sample is obtained and the assay is performed within said 24 hourperiod.

Section headings as used in this section and the entire disclosureherein are merely for organizational purposes and are not intended to belimiting.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity matured antibody” is used herein to refer to an antibody withone or more alterations in one or more CDRs, which result in animprovement in the affinity (i.e., K_(D), k_(d) or k_(a)) of theantibody for a target antigen compared to a parent antibody, which doesnot possess the alteration(s). Exemplary affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.A variety of procedures for producing affinity matured antibodies isknown in the art, including the screening of a combinatory antibodylibrary that has been prepared using bio-display. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); and Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity-enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

“Antibody” and “antibodies” as used herein refers to monoclonalantibodies, multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized), animal antibodies such as,but not limited to, a bird (for example, a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate(for example, a monkey, a chimpanzee, etc.), recombinant antibodies,chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments, F(ab′)₂fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11):1290-1297 (2007) and PCT International ApplicationWO 2001/058956, the contents of each of which are herein incorporated byreference), and functionally active epitope-binding fragments of any ofthe above. In particular, antibodies include immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules,namely, molecules that contain an analyte-binding site. Immunoglobulinmolecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, andIgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), orsubclass. For simplicity sake, an antibody against an analyte isfrequently referred to herein as being either an “anti-analyte antibody”or merely an “analyte antibody” (e.g., an anti-UCH-L1 antibody or aUCH-L1 antibody).

“Antibody fragment” as used herein refers to a portion of an intactantibody comprising the antigen-binding site or variable region. Theportion does not include the constant heavy chain domains (i.e., CH2,CH3, or CH4, depending on the antibody isotype) of the Fc region of theintact antibody. Examples of antibody fragments include, but are notlimited to, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)₂fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv)molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs ofthe light-chain variable domain, single-chain polypeptides containingonly one heavy chain variable region, and single-chain polypeptidescontaining the three CDRs of the heavy chain variable region.

The “area under curve” or “AUC” refers to area under a ROC curve. AUCunder a ROC curve is a measure of accuracy. An AUC of 1 represents aperfect test, whereas an AUC of 0.5 represents an insignificant test. Apreferred AUC may be at least approximately 0.700, at leastapproximately 0.750, at least approximately 0.800, at leastapproximately 0.850; at least approximately 0.900, at leastapproximately 0.910, at least approximately 0.920, at leastapproximately 0.930, at least approximately 0.940, at leastapproximately 0.950, at least approximately 0.960, at leastapproximately 0.970, at least approximately 0.980, at leastapproximately 0.990, or at least approximately 0.995.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support. One example of a bead or particleis a microparticle. Microparticles that can be used herein can be anytype known in the art. For example, the bead or particle can be amagnetic bead or magnetic particle. Magnetic beads/particles may beferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄(or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The microparticles can be of any size thatwould work in the methods described herein, e.g., from about 0.75 toabout 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3nm.

“Binding protein” is used herein to refer to a monomeric or multimericprotein that binds to and forms a complex with a binding partner, suchas, for example, a polypeptide, an antigen, a chemical compound or othermolecule, or a substrate of any kind. A binding protein specificallybinds a binding partner. Binding proteins include antibodies, as well asantigen-binding fragments thereof and other various forms andderivatives thereof as are known in the art and described herein below,and other molecules comprising one or more antigen-binding domains thatbind to an antigen molecule or a particular site (epitope) on theantigen molecule. Accordingly, a binding protein includes, but is notlimited to, an antibody a tetrameric immunoglobulin, an IgG molecule, anIgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

“Bispecific antibody” is used herein to refer to a full-length antibodythat is generated by quadroma technology (see Milstein et al., Nature,305(5934): 537-540 (1983)), by chemical conjugation of two differentmonoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628-631(1985)), or by knob-into-hole or similar approaches, which introducemutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci.USA, 90(14): 6444-6448 (1993)), resulting in multiple differentimmunoglobulin species of which only one is the functional bispecificantibody. A bispecific antibody binds one antigen (or epitope) on one ofits two binding arms (one pair of HC/LC), and binds a different antigen(or epitope) on its second arm (a different pair of HC/LC). By thisdefinition, a bispecific antibody has two distinct antigen-binding arms(in both specificity and CDR sequences), and is monovalent for eachantigen to which it binds to.

“CDR” is used herein to refer to the “complementarity determiningregion” within an antibody variable sequence. There are three CDRs ineach of the variable regions of the heavy chain and the light chain.Proceeding from the N-terminus of a heavy or light chain, these regionsare denoted “CDR1”, “CDR2”, and “CDR3”, for each of the variableregions. The term “CDR set” as used herein refers to a group of threeCDRs that occur in a single variable region that binds the antigen. Anantigen-binding site, therefore, may include six CDRs, comprising theCDR set from each of a heavy and a light chain variable region. Apolypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) maybe referred to as a “molecular recognition unit.” Crystallographicanalyses of antigen-antibody complexes have demonstrated that the aminoacid residues of CDRs form extensive contact with bound antigen, whereinthe most extensive antigen contact is with the heavy chain CDR3. Thus,the molecular recognition units may be primarily responsible for thespecificity of an antigen-binding site. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as “Kabat CDRs”.Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917(1987); and Chothia et al., Nature, 342: 877-883 (1989)) found thatcertain sub-portions within Kabat CDRs adopt nearly identical peptidebackbone conformations, despite having great diversity at the level ofamino acid sequence. These sub-portions were designated as “L1”, “L2”,and “L3”, or “H1”, “H2”, and “H3”, where the “L” and the “H” designatethe light chain and the heavy chain regions, respectively. These regionsmay be referred to as “Chothia CDRs”, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995),and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDRboundary definitions may not strictly follow one of the herein systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat- or Chothia-defined CDRs.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection or conjugate a calibrator, a control,a sensitivity panel, a container, a buffer, a diluent, a salt, anenzyme, a co-factor for an enzyme, a detection reagent, a pretreatmentreagent/solution, a substrate (e.g., as a solution), a stop solution,and the like that can be included in a kit for assay of a test sample,such as a patient urine, whole blood, serum or plasma sample, inaccordance with the methods described herein and other methods known inthe art. Some components can be in solution or lyophilized forreconstitution for use in an assay.

“Correlated to” as used herein refers to compared to.

“CT scan” as used herein refers to a computerized tomography (CT) scan.A CT scan combines a series of X-ray images taken from different anglesand uses computer processing to create cross-sectional images, orslices, of the bones, blood vessels and soft tissues inside your body.The CT scan may use X-ray CT, positron emission tomography (PET),single-photon emission computed tomography (SPECT), computed axialtomography (CAT scan), or computer aided tomography. The CT scan may bea conventional CT scan or a spiral/helical CT scan. In a conventional CTscan, the scan is taken slice by slice and after each slice the scanstops and moves down to the next slice, e.g., from the top of theabdomen down to the pelvis. The conventional CT scan requires patientsto hold their breath to avoid movement artefact. The spiral/helical CTscan is a continuous scan which is taken in a spiral fashion and is amuch quicker process where the scanned images are contiguous.

“Determined by an assay” is used herein to refer to the determination ofa reference level by any appropriate assay. The determination of areference level may, in some embodiments, be achieved by an assay of thesame type as the assay that is to be applied to the sample from thesubject (for example, by an immunoassay, clinical chemistry assay, asingle molecule detection assay, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS)). The determination of a reference level may, in someembodiments, be achieved by an assay of the same type and under the sameassay conditions as the assay that is to be applied to the sample fromthe subject. As noted herein, this disclosure provides exemplaryreference levels (e.g., calculated by comparing reference levels atdifferent time points). It is well within the ordinary skill of one inthe art to adapt the disclosure herein for other assays to obtainassay-specific reference levels for those other assays based on thedescription provided by this disclosure. For example, a set of trainingsamples comprising samples obtained from human subjects known to havesustained an injury to the head (and more particularly, samples obtainedfrom human subjects known to have sustained a (i) mild TBI; and/or (ii)moderate, severe, or moderate to severe TBI and samples obtained fromhuman subjects known not to have sustained an injury to the head may beused to obtain assay-specific reference levels. It will be understoodthat a reference level “determined by an assay” and having a recitedlevel of “sensitivity” and/or “specificity” is used herein to refer to areference level which has been determined to provide a method of therecited sensitivity and/or specificity when said reference level isadopted in the methods of the invention. It is well within the ordinaryskill of one in the art to determine the sensitivity and specificityassociated with a given reference level in the methods of the invention,for example by repeated statistical analysis of assay data using aplurality of different possible reference levels.

Practically, when discriminating between a subject as having a traumaticbrain injury or not having a traumatic brain injury or a subject ashaving a a mild versus a moderate, severe, or moderate to severetraumatic brain injury, the skilled person will balance the effect ofraising a cutoff on sensitivity and specificity. Raising or lowering acutoff will have a well-defined and predictable impact on sensitivityand specificity, and other standard statistical measures. It is wellknown that raising a cutoff will improve specificity but is likely toworsen sensitivity (proportion of those with disease who test positive).In contrast, lowering a cutoff will improve sensitivity but will worsenspecificity (proportion of those without disease who test negative). Theramifications for detecting traumatic brain injury or determining a mildversus moderate, severe, or moderate to severe traumatic brain injurywill be readily apparent to those skilled in the art. In discriminatingwhether a subject has or does not have a traumatic brain injury or amild versus a moderate, severe, or moderate to severe traumatic braininjury, the higher the cutoff, specificity improves as more truenegatives (i.e., subjects not having a traumatic brain injury, nothaving a mild traumatic brain injury, not have a moderate traumaticbrain injury, not having a severe traumatic brain injury or not having amoderate to severe traumatic brain injury) are distinguished from thosehaving a traumatic brain injury, a mild traumatic brain injury, amoderate traumatic brain injury, a severe traumatic brain injury or amoderate to severe traumatic brain injury. But at the same time, raisingthe cutoff decreases the number of cases identified as positive overall,as well as the number of true positives, so the sensitivity mustdecrease. Conversely, the lower the cutoff, sensitivity improves as moretrue positives (i.e., subjects having a traumatic brain injury, having amild traumatic brain injury, having a moderate traumatic brain injury,having a severe traumatic brain injury or having a moderate to severetraumatic brain injury) are distinguished from those who do not have atraumatic brain injury, a mild traumatic brain injure, a moderatetraumatic brain injury, a severe traumatic brain injury or a moderate tosevere traumatic brain injury. But at the same time, lowering the cutoffincreases the number of cases identified as positive overall, as well asthe number of false positives, so the specificity must decrease.

Generally, a high sensitivity value helps one of skill rule out diseaseor condition (such as a traumatic brain injury, mild traumatic braininjury, moderate traumatic brain injury, severe traumatic brain injuryor moderate to severe traumatic brain injury), and a high specificityvalue helps one of skill rule in disease or condition. Whether one ofskill desires to rule out or rule in disease depends on what theconsequences are for the patient for each type of error. Accordingly,one cannot know or predict the precise balancing employed to derive atest cutoff without full disclosure of the underlying information on howthe value was selected. The balancing of sensitivity against specificityand other factors-will differ on a case-by-case basis. This is why it issometimes preferable to provide alternate cutoff (e.g., reference)values so a physician or practitioner can choose.

“Derivative” of an antibody as used herein may refer to an antibodyhaving one or more modifications to its amino acid sequence whencompared to a genuine or parent antibody and exhibit a modified domainstructure. The derivative may still be able to adopt the typical domainconfiguration found in native antibodies, as well as an amino acidsequence, which is able to bind to targets (antigens) with specificity.Typical examples of antibody derivatives are antibodies coupled to otherpolypeptides, rearranged antibody domains, or fragments of antibodies.The derivative may also comprise at least one further compound, e.g., aprotein domain, said protein domain being linked by covalent ornon-covalent bonds. The linkage can be based on genetic fusion accordingto the methods known in the art. The additional domain present in thefusion protein comprising the antibody may preferably be linked by aflexible linker, advantageously a peptide linker, wherein said peptidelinker comprises plural, hydrophilic, peptide-bonded amino acids of alength sufficient to span the distance between the C-terminal end of thefurther protein domain and the N-terminal end of the antibody or viceversa. The antibody may be linked to an effector molecule having aconformation suitable for biological activity or selective binding to asolid support, a biologically active substance (e.g., a cytokine orgrowth hormone), a chemical agent, a peptide, a protein, or a drug, forexample.

“Drugs of abuse” is used herein to refer to one or more additivesubstances (such as a drug) taken for non-medical reasons (such as for,example, recreational and/or mind-altering effects). Excessiveoverindulgence, use or dependence of such drugs of abuse is oftenreferred to as “substance abuse”. Examples of drugs of abuse includealcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens(such as ketamine, mescaline (peyote), PCP, psilocybin, DMT and/or LSD),methaqualone, opioids, amphetamines (including methamphetamines),anabolic steroids, inhalants (namely, substances which contain volatilesubstances that contain psychoactive properties such as, for example,nitrites, spray paints, cleaning fluids, markers, glues, etc.) andcombinations thereof.

“Dual-specific antibody” is used herein to refer to a full-lengthantibody that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly, a dual-specific binding protein has twoidentical antigen binding arms, with identical specificity and identicalCDR sequences, and is bivalent for each antigen to which it binds.

“Dual variable domain” is used herein to refer to two or more antigenbinding sites on a binding protein, which may be divalent (two antigenbinding sites), tetravalent (four antigen binding sites), or multivalentbinding proteins. DVDs may be monospecific, i.e., capable of binding oneantigen (or one specific epitope), or multispecific, i.e., capable ofbinding two or more antigens (i.e., two or more epitopes of the sametarget antigen molecule or two or more epitopes of different targetantigens). A preferred DVD binding protein comprises two heavy chain DVDpolypeptides and two light chain DVD polypeptides and is referred to asa “DVD immunoglobulin” or “DVD-Ig.” Such a DVD-Ig binding protein isthus tetrameric and reminiscent of an IgG molecule, but provides moreantigen binding sites than an IgG molecule. Thus, each half of atetrameric DVD-Ig molecule is reminiscent of one half of an IgG moleculeand comprises a heavy chain DVD polypeptide and a light chain DVDpolypeptide, but unlike a pair of heavy and light chains of an IgGmolecule that provides a single antigen binding domain, a pair of heavyand light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of a DVD-Ig binding protein may be derivedfrom a donor (“parental”) monoclonal antibody and thus comprises a heavychain variable domain (VH) and a light chain variable domain (VL) with atotal of six CDRs involved in antigen binding per antigen binding site.Accordingly, a DVD-Ig binding protein that binds two different epitopes(i.e., two different epitopes of two different antigen molecules or twodifferent epitopes of the same antigen molecule) comprises an antigenbinding site derived from a first parental monoclonal antibody and anantigen binding site of a second parental monoclonal antibody.

A description of the design, expression, and characterization of DVD-Igbinding molecules is provided in PCT Publication No. WO 2007/024715,U.S. Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297(2007). A preferred example of such DVD-Ig molecules comprises a heavychain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a heavy chain constant domain, X1 is alinker with the proviso that it is not CH1, X2 is an Fc region, and n is0 or 1, but preferably 1; and a light chain that comprises thestructural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain, X1 is a linker with the proviso that itis not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, butpreferably 1. Such a DVD-Ig may comprise two such heavy chains and twosuch light chains, wherein each chain comprises variable domains linkedin tandem without an intervening constant region between variableregions, wherein a heavy chain and a light chain associate to formtandem functional antigen binding sites, and a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1, VD2, VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate to formthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein not only binds thesame target molecules bound by its parental monoclonal antibodies, butalso possesses one or more desirable properties of one or more of itsparental monoclonal antibodies. Preferably, such an additional propertyis an antibody parameter of one or more of the parental monoclonalantibodies. Antibody parameters that may be contributed to a DVD-Igbinding protein from one or more of its parental monoclonal antibodiesinclude, but are not limited to, antigen specificity, antigen affinity,potency, biological function, epitope recognition, protein stability,protein solubility, production efficiency, immunogenicity,pharmacokinetics, bioavailability, tissue cross reactivity, andorthologous antigen binding.

A DVD-Ig binding protein binds at least one epitope of UCH-L1.Non-limiting examples of a DVD-Ig binding protein include a DVD-Igbinding protein that binds one or more epitopes of UCH-L1, a DVD-Igbinding protein that binds an epitope of a human UCH-L1 and an epitopeof UCH-L1 of another species (for example, mouse), and a DVD-Ig bindingprotein that binds an epitope of a human UCH-L1 and an epitope ofanother target molecule.

“Dynamic range” as used herein refers to range over which an assayreadout is proportional to the amount of target molecule or analyte inthe sample being analyzed.

“Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s)on any molecule that is recognized and can bind to a complementarysite(s) on its specific binding partner. The molecule and specificbinding partner are part of a specific binding pair. For example, anepitope can be on a polypeptide, a protein, a hapten, a carbohydrateantigen (such as, but not limited to, glycolipids, glycoproteins orlipopolysaccharides), or a polysaccharide. Its specific binding partnercan be, but is not limited to, an antibody.

“Fragment antigen-binding fragment” or “Fab fragment” as used hereinrefers to a fragment of an antibody that binds to antigens and thatcontains one antigen-binding site, one complete light chain, and part ofone heavy chain. Fab is a monovalent fragment consisting of the VL, VH,CL and CH1 domains. Fab is composed of one constant and one variabledomain of each of the heavy and the light chain. The variable domaincontains the paratope (the antigen-binding site), comprising a set ofcomplementarity determining regions, at the amino terminal end of themonomer. Each arm of the Y thus binds an epitope on the antigen. Fabfragments can be generated such as has been described in the art, e.g.,using the enzyme papain, which can be used to cleave an immunoglobulinmonomer into two Fab fragments and an Fc fragment, or can be produced byrecombinant means.

“F(ab′)₂ fragment” as used herein refers to antibodies generated bypepsin digestion of whole IgG antibodies to remove most of the Fc regionwhile leaving intact some of the hinge region. F(ab′)₂ fragments havetwo antigen-binding F(ab) portions linked together by disulfide bonds,and therefore are divalent with a molecular weight of about 110 kDa.Divalent antibody fragments (F(ab′)₂ fragments) are smaller than wholeIgG molecules and enable a better penetration into tissue thusfacilitating better antigen recognition in immunohistochemistry. The useof F(ab′)₂ fragments also avoids unspecific binding to Fc receptor onlive cells or to Protein A/G. F(ab′)₂ fragments can both bind andprecipitate antigens.

“Framework” (FR) or “Framework sequence” as used herein may mean theremaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems (for example, see above), the meaning of a framework sequence issubject to correspondingly different interpretations. The six CDRs(CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavychain) also divide the framework regions on the light chain and theheavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on eachchain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2and FR3, and CDR3 between FR3 and FR4. Without specifying the particularsub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred byothers, represents the combined FRs within the variable region of asingle, naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain FR sequences are known in the art thatcan be used as heavy chain and light chain “acceptor” frameworksequences (or simply, “acceptor” sequences) to humanize a non-humanantibody using techniques known in the art. In one embodiment, humanheavy chain and light chain acceptor sequences are selected from theframework sequences listed in publicly available databases such asV-base (hypertext transfer protocol://vbase.mrc-cpe.cam.ac.uk/) or inthe international ImMunoGeneTics® (IMGT®) information system (hypertexttransfer protocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).

“Functional antigen binding site” as used herein may mean a site on abinding protein (e.g., an antibody) that is capable of binding a targetantigen. The antigen binding affinity of the antigen binding site maynot be as strong as the parent binding protein, e.g., parent antibody,from which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating protein, e.g., antibody, binding to an antigen. Moreover,the antigen binding affinity of each of the antigen binding sites of amultivalent protein, e.g., multivalent antibody, herein need not bequantitatively the same.

“GFAP” is used herein to describe glial fibrillary acidic protein. GFAPis a protein that is encoded by the GFAP gene in humans, and which canbe produced (e.g., by recombinant means, in other species).

“GFAP status” can mean either the level or amount of GFAP at a point intime (such as with a single measure of GFAP), the level or amount ofGFAP associated with monitoring (such as with a repeat test on a subjectto identify an increase or decrease in GFAP amount), the level or amountof GFAP associated with treatment for traumatic brain injury (whether aprimary brain injury and/or a secondary brain injury) or combinationsthereof.

“Glasgow Coma Scale” or “GCS” as used herein refers to a 15 point scalefor estimating and categorizing the outcomes of brain injury on thebasis of overall social capability or dependence on others. The testmeasures the motor response, verbal response and eye opening responsewith these values: I. Motor Response (6—Obeys commands fully;5—Localizes to noxious stimuli; 4—Withdraws from noxious stimuli;3—Abnormal flexion, i.e., decorticate posturing; 2—Extensor response,i.e., decerebrate posturing; and 1—No response); II. Verbal Response(5—Alert and Oriented; 4—Confused, yet coherent, speech; 3—Inappropriatewords and jumbled phrases consisting of words; 2—Incomprehensiblesounds; and 1—No sounds); and III. Eye Opening (4—Spontaneous eyeopening; 3—Eyes open to speech; 2—Eyes open to pain; and 1—No eyeopening). The final score is determined by adding the values ofI+II+III. The final score can be categorized into four possible levelsfor survival, with a lower number indicating a more severe injury and apoorer prognosis: Mild (13-15); Moderate Disability (9-12) (Loss ofconsciousness greater than 30 minutes; Physical or cognitive impairmentswhich may or may resolve: and Benefit from Rehabilitation); SevereDisability (3-8) (Coma: unconscious state. No meaningful response, novoluntary activities); and Vegetative State (Less Than 3) (Sleep wakecycles; Arousal, but no interaction with environment; No localizedresponse to pain). Moderate brain injury is defined as a brain injuryresulting in a loss of consciousness from 20 minutes to 6 hours and aGlasgow Coma Scale of 9 to 12. Severe brain injury is defined as a braininjury resulting in a loss of consciousness of greater than 6 hours anda Glasgow Coma Scale of 3 to 8.

“Glasgow Outcome Scale” as used herein refers to a global scale forfunctional outcome that rates patient status into one of fivecategories: Dead, Vegetative State, Severe Disability, ModerateDisability or Good Recovery.

“Extended Glasgow Outcome Scale” or “GOSE” as used interchangeablyherein provides more detailed categorization into eight categories bysubdividing the categories of severe disability, moderate disability andgood recovery into a lower and upper category as shown in Table 1.

TABLE 1 1 Death D 2 Vegetative state VX 3 Lower severe disability SD−Condition of unawareness with only reflex 4 Upper severe disability SD+responses but with periods of spontaneous eye opening 5 Lower moderatedisability MD− Patient who is dependent for daily support for 6 Uppermoderate disability MD+ mental or physical disability, usually acombination of both. If the patient can be left alone for more than 8hours at home it is upper level of SD, if not then it is low level ofSD. 7 Lower good recovery GR− Patients have some disability such asaphasia, 8 Upper good recovery GR+ hemiparesis or epilepsy and/ordeficits of memory or personality but are able to look after themselves.They are independent at home but dependent outside. If they are able toreturn to work even with special arrangement it is upper level of MD, ifnot then it is low level of MD.

“Humanized antibody” is used herein to describe an antibody thatcomprises heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or VL sequence has been altered to be more “human-like,” i.e.,more similar to human germline variable sequences. A “humanizedantibody” is an antibody or a variant, derivative, analog, or fragmentthereof, which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of a non-human antibody CDR. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. In an embodiment, a humanized antibody also comprises at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsthe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

A humanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE, and any isotype, includingwithout limitation IgG1, IgG2, IgG3, and IgG4. A humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well-known in the art.

The framework regions and CDRs of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion, and/or deletion of at least one amino acid residue so thatthe CDR or framework residue at that site does not correspond to eitherthe donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence.

“Identical” or “identity,” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are the same over aspecified region. The percentage can be calculated by optimally aligningthe two sequences, comparing the two sequences over the specifiedregion, determining the number of positions at which the identicalresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the specified region, and multiplying the result by 100to yield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of the single sequence are included in thedenominator but not the numerator of the calculation.

“Imaging procedure” as used herein refers to a medical test that allowsthe inside of a body to be seen in order to diagnose, treat, and monitorhealth conditions. An imaging procedure can be a non-invasive procedurethat allows diagnosis of diseases and injuries without being intrusive.Examples of imaging procedures include MRI, CT scan, X-rays, positronemission tomography (PET) scan, single-photon emission computedtomography (SPECT), and diffusion tensor imaging (DTI) scan.

“Injury to the head” or “head injury” as used interchangeably herein,refers to any trauma to the scalp, skull, or brain. Such injuries mayinclude only a minor bump on the skull or may be a serious brain injury.Such injuries include primary injuries to the brain and/or secondaryinjuries to the brain. Primary brain injuries occur during the initialinsult and result from displacement of the physical structures of thebrain. More specifically, a primary brain injury is the physical damageto parenchyma (tissue, vessels) that occurs during the traumatic event,resulting in shearing and compression of the surrounding brain tissue.Secondary brain injuries occur subsequent to the primary injury and mayinvolve an array of cellular processes. More specifically, a secondarybrain injury refers to the changes that evolve over a period of time(from hours to days) after the primary brain injury. It includes anentire cascade of cellular, chemical, tissue, or blood vessel changes inthe brain that contribute to further destruction of brain tissue.

An injury to the head can be either closed or open (penetrating). Aclosed head injury refers to a trauma to the scalp, skull or brain wherethere is no penetration of the skull by a striking object. An open headinjury refers a trauma to the scalp, skull or brain where there ispenetration of the skull by a striking object. An injury to the head maybe caused by physical shaking of a person, by blunt impact by anexternal mechanical or other force that results in a closed or open headtrauma (e.g., vehicle accident such as with an automobile, plane, train,etc.; blow to the head such as with a baseball bat, or from a firearm),a cerebral vascular accident (e.g., stroke), one or more falls (e.g., asin sports or other activities), explosions or blasts (collectively,“blast injuries”) and by other types of blunt force trauma.Alternatively, an injury to the head may be caused by the ingestionand/or exposure to a chemical, toxin or a combination of a chemical andtoxin. Examples of such chemicals and/or toxins include fires, molds,asbestos, pesticides and insecticides, organic solvents, paints, glues,gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organicmetals (such as methyl mercury, tetraethyl lead and organic tin) and/orone or more drugs of abuse. Alternatively, an injury to the head may becaused as a result of a subject suffering from an autoimmune disease, ametabolic disorder, a brain tumor, one or more viruses, meningitis,hydrocephalus, hypoxia or any combinations thereof. In some cases, it isnot possible to be certain whether any such event or injury has occurredor taken place. For example, there may be no history on a patient orsubject, the subject may be unable to speak, the subject may be aware ofwhat events they were exposed to, etc. Such circumstances are describedherein as the subject “may have sustained an injury to the head.” Incertain embodiments herein, the closed head injury does not include andspecifically excludes a cerebral vascular accident, such as stroke.

“Intracranial lesion” as used herein refers to an area of injury withinthe brain. An intracranial lesion can be an abnormality seen on aimaging procedure or brain-imaging test, such as MRI or CT scan. On CTor MRI scans, brain lesions can appear as dark or light spots that donot look like normal brain tissue.

“Isolated polynucleotide” as used herein may mean a polynucleotide(e.g., of genomic, cDNA, or synthetic origin, or a combination thereof)that, by virtue of its origin, the isolated polynucleotide is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

“Label” and “detectable label” as used herein refer to a moiety attachedto an antibody or an analyte to render the reaction between the antibodyand the analyte detectable, and the antibody or analyte so labeled isreferred to as “detectably labeled.” A label can produce a signal thatis detectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromagens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like.Representative examples of labels include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety, itself, may not be detectable but may become detectable uponreaction with yet another moiety. Use of the term “detectably labeled”is intended to encompass such labeling.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as 3H,14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and153Sm), an enzymatic label (such as horseradish peroxidase, alkalineperoxidase, glucose 6-phosphate dehydrogenase, and the like), achemiluminescent label (such as acridinium esters, thioesters, orsulfonamides; luminol, isoluminol, phenanthridinium esters, and thelike), a fluorescent label (such as fluorescein (e.g., 5-fluorescein,6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein,6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluoresceinisothiocyanate, and the like)), rhodamine, phycobiliproteins,R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmiumselenide), a thermometric label, or an immuno-polymerase chain reactionlabel. An introduction to labels, labeling procedures and detection oflabels is found in Polak and Van Noorden, Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and inHaugland, Handbook of Fluorescent Probes and Research Chemicals (1996),which is a combined handbook and catalogue published by MolecularProbes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA(see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,and 5,352,803, which are hereby incorporated by reference in theirentireties). An acridinium compound can be used as a detectable label ina homogeneous chemiluminescent assay (see, e.g., Adamczyk et al.,Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg.Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem.Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782(2003)).

In one aspect, the acridinium compound is an acridinium-9-carboxamide.Methods for preparing acridinium 9-carboxamides are described inMattingly, J Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., JOrg. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999);Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly etal., In Luminescence Biotechnology: Instruments and Applications; Dyke,K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524and 5,783,699 (each of which is incorporated herein by reference in itsentirety for its teachings regarding same).

Another example of an acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

“Linking sequence” or “linking peptide sequence” refers to a natural orartificial polypeptide sequence that is connected to one or morepolypeptide sequences of interest (e.g., full-length, fragments, etc.).The term “connected” refers to the joining of the linking sequence tothe polypeptide sequence of interest. Such polypeptide sequences arepreferably joined by one or more peptide bonds. Linking sequences canhave a length of from about 4 to about 50 amino acids. Preferably, thelength of the linking sequence is from about 6 to about 30 amino acids.Natural linking sequences can be modified by amino acid substitutions,additions, or deletions to create artificial linking sequences. Linkingsequences can be used for many purposes, including in recombinant Fabs.Exemplary linking sequences include, but are not limited to: (i)Histidine (His) tags, such as a 6×His tag, which has an amino acidsequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences tofacilitate the isolation and purification of polypeptides and antibodiesof interest; (ii) Enterokinase cleavage sites, like His tags, are usedin the isolation and purification of proteins and antibodies ofinterest. Often, enterokinase cleavage sites are used together with Histags in the isolation and purification of proteins and antibodies ofinterest. Various enterokinase cleavage sites are known in the art.Examples of enterokinase cleavage sites include, but are not limited to,the amino acid sequence of DDDDK (SEQ ID NO: 4) and derivatives thereof(e.g., ADDDDK (SEQ ID NO: 5), etc.); (iii) Miscellaneous sequences canbe used to link or connect the light and/or heavy chain variable regionsof single chain variable region fragments. Examples of other linkingsequences can be found in Bird et al., Science 242: 423-426 (1988);Huston et al., PNAS USA 85: 5879-5883 (1988); and McCafferty et al.,Nature 348: 552-554 (1990). Linking sequences also can be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. In the context of the present disclosure, the monoclonalantibody, for example, can contain a linking sequence, such as a Histag, an enterokinase cleavage site, or both.

“Magnetic resonance imaging” or “MRI” as used interchangeably hereinrefers to a medical imaging technique used in radiology to form picturesof the anatomy and the physiological processes of the body in bothhealth and disease (e.g., referred to herein interchangeably as “anMRI”, “an MRI procedure” or “an MRI scan”). MRI is a form of medicalimaging that measures the response of the atomic nuclei of body tissuesto high-frequency radio waves when placed in a strong magnetic field,and that produces images of the internal organs. MRI scanners, which isbased on the science of nuclear magnetic resonance (NMR), use strongmagnetic fields, radio waves, and field gradients to generate images ofthe inside of the body.

“Monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological.

“Multivalent binding protein” is used herein to refer to a bindingprotein comprising two or more antigen binding sites (also referred toherein as “antigen binding domains”). A multivalent binding protein ispreferably engineered to have three or more antigen binding sites, andis generally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein that can bind two or morerelated or unrelated targets, including a binding protein capable ofbinding two or more different epitopes of the same target molecule.

“Negative predictive value” or “NPV” as used interchangeably hereinrefers to the probability that a subject has a negative outcome giventhat they have a negative test result.

“Reference level” as used herein refers to an assay cutoff value that isused to assess diagnostic, prognostic, or therapeutic efficacy and thathas been linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.). An“absolute amount” as used herein refers to the absolute value of achange or difference between at least two assay results taken or sampledat different time points and, which similar to a reference level, hasbeen linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.).“Absolute value” as used herein refers to the magnitude of a real number(such as, for example, the difference between two compared levels (suchas levels taken at a first time point and levels taken at a second timepoint)) without regard to its sign, i.e., regardless of whether it ispositive or negative.

This disclosure provides exemplary reference levels and absolute amounts(e.g., calculated by comparing reference levels at different timepoints). However, it is well-known that reference levels and absoluteamounts may vary depending on the nature of the immunoassay (e.g.,antibodies employed, reaction conditions, sample purity, etc.) and thatassays can be compared and standardized. It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific reference levels andabsolute amounts for those other immunoassays based on the descriptionprovided by this disclosure. Whereas the precise value of the referencelevel and absolute amount may vary between assays, the findings asdescribed herein should be generally applicable and capable of beingextrapolated to other assays.

“Point-of-care device” refers to a device used to provide medicaldiagnostic testing at or near the point-of-care (namely, outside of alaboratory), at the time and place of patient care (such as in ahospital, physician's office, urgent or other medical care facility, apatient's home, a nursing home and/or a long term care and/or hospicefacility). Examples of point-of-care devices include those produced byAbbott Laboratories (Abbott Park, Ill.) (e.g., i-STAT and i-STATAlinity, Universal Biosensors (Rowville, Australia) (see US2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical LabProducts (Los Angeles, USA).

“Positive predictive value” or “PPV” as used interchangeably hereinrefers to the probability that a subject has a positive outcome giventhat they have a positive test result.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a reference level or control level (e.g.,“low”, “medium”, or “high” levels), can be used. Multiple calibrators(i.e., more than one calibrator or a varying amount of calibrator(s))can be used in conjunction to comprise a “sensitivity panel.”

A “receiver operating characteristic” curve or “ROC” curve refers to agraphical plot that illustrates the performance of a binary classifiersystem as its discrimination threshold is varied. For example, an ROCcurve can be a plot of the true positive rate against the false positiverate for the different possible cutoff points of a diagnostic test. Itis created by plotting the fraction of true positives out of thepositives (TPR=true positive rate) vs. the fraction of false positivesout of the negatives (FPR=false positive rate), at various thresholdsettings. TPR is also known as sensitivity, and FPR is one minus thespecificity or true negative rate. The ROC curve demonstrates thetradeoff between sensitivity and specificity (any increase insensitivity will be accompanied by a decrease in specificity); thecloser the curve follows the left-hand border and then the top border ofthe ROC space, the more accurate the test; the closer the curve comes tothe 45-degree diagonal of the ROC space, the less accurate the test; theslope of the tangent line at a cutoff point gives the likelihood ratio(LR) for that value of the test; and the area under the curve is ameasure of test accuracy.

“Recombinant antibody” and “recombinant antibodies” refer to antibodiesprepared by one or more steps, including cloning nucleic acid sequencesencoding all or a part of one or more monoclonal antibodies into anappropriate expression vector by recombinant techniques and subsequentlyexpressing the antibody in an appropriate host cell. The terms include,but are not limited to, recombinantly produced monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multi-specific or multi-valent structures formed fromantibody fragments, bifunctional antibodies, heteroconjugate Abs,DVD-Ig®s, and other antibodies as described in (i) herein.(Dual-variable domain immunoglobulins and methods for making them aredescribed in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

“Risk assessment,” “risk classification,” “risk identification,” or“risk stratification” of subjects (e.g., patients) as used herein refersto the evaluation of factors including biomarkers, to predict the riskof occurrence of future events including disease onset or diseaseprogression, so that treatment decisions regarding the subject may bemade on a more informed basis.

“Sample,” “test sample,” “specimen,” “sample from a subject,” and“patient sample” as used herein may be used interchangeable and may be asample of blood such as whole blood, tissue, urine, serum, plasma,amniotic fluid, cerebrospinal fluid, placental cells or tissue,endothelial cells, leukocytes, or monocytes. In some embodiments, thesample is a whole blood sample. In some embodiments, the sample is aserum sample. In yet other embodiments, the sample is a plasma sample.The sample can be used directly as obtained from a patient or can bepre-treated, such as by filtration, distillation, extraction,concentration, centrifugation, inactivation of interfering components,addition of reagents, and the like, to modify the character of thesample in some manner as discussed herein or otherwise as is known inthe art.

A variety of cell types, tissue, or bodily fluid may be utilized toobtain a sample. Such cell types, tissues, and fluid may includesections of tissues such as biopsy and autopsy samples, frozen sectionstaken for histologic purposes, blood (such as whole blood), plasma,serum, red blood cells, platelets, interstitial fluid, cerebral spinalfluid, etc. Cell types and tissues may also include lymph fluid,cerebrospinal fluid, a fluid collected by A tissue or cell type may beprovided by removing a sample of cells from a human and a non-humananimal, but can also be accomplished by using previously isolated cells(e.g., isolated by another person, at another time, and/or for anotherpurpose). Archival tissues, such as those having treatment or outcomehistory, may also be used. Protein or nucleotide isolation and/orpurification may not be necessary.

“Sensitivity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is positive that are correctly identifiedas positive (e.g., correctly identifying those subjects with a diseaseor medical condition for which they are being tested). For example, thismight include correctly identifying subjects as having a TBI from thosewho do not have a TBI, correctly identifying subjects having a moderate,severe, or moderate to severe TBI from those having a mild TBI,correctly identifying subjects as having a mild TBI from those having amoderate, severe, or moderate to severe TBI, correctly identifyingsubjects as having a moderate, severe, or moderate to severe TBI fromthose having no TBI or correctly identifying subjects as having a mildTBI from those having no TBI, correctly identifying subjects as likelyto benefit from imaging or a head CT scan or a MRI from those who arenot likely to benefit from a head imaging or a CT scan or MRI, etc.).

“Specificity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is negative that are correctly identifiedas negative (e.g., correctly identifying those subjects who do not havea disease or medical condition for which they are being tested). Forexample, this might include correctly identifying subjects having an TBIfrom those who do not have a TBI, correctly identifying subjects nothaving a moderate, severe, or moderate to severe TBI from those having amild TBI, correctly identifying subjects as not having a mild TBI fromthose having a moderate, severe, or moderate to severe TBI or correctlyidentifying subjects as not having any TBI, or correctly identifyingsubjects as having a mild TBI from those having no TBI, etc.).

“Series of calibrating compositions” refers to a plurality ofcompositions comprising a known concentration of UCH-L1, wherein each ofthe compositions differs from the other compositions in the series bythe concentration of UCH-L1.

“Solid phase” or “solid support” as used interchangeably herein, refersto any material that can be used to attach and/or attract and immobilize(1) one or more capture agents or capture specific binding partners, or(2) one or more detection agents or detection specific binding partners.The solid phase can be chosen for its intrinsic ability to attract andimmobilize a capture agent. Alternatively, the solid phase can haveaffixed thereto a linking agent that has the ability to attract andimmobilize the (1) capture agent or capture specific binding partner, or(2) detection agent or detection specific binding partner. For example,the linking agent can include a charged substance that is oppositelycharged with respect to the capture agent (e.g., capture specificbinding partner) or detection agent (e.g., detection specific bindingpartner) itself or to a charged substance conjugated to the (1) captureagent or capture specific binding partner or (2) detection agent ordetection specific binding partner. In general, the linking agent can beany binding partner (preferably specific) that is immobilized on(attached to) the solid phase and that has the ability to immobilize the(1) capture agent or capture specific binding partner, or (2) detectionagent or detection specific binding partner through a binding reaction.The linking agent enables the indirect binding of the capture agent to asolid phase material before the performance of the assay or during theperformance of the assay. For examples, the solid phase can be plastic,derivatized plastic, magnetic, or non-magnetic metal, glass or silicon,including, for example, a test tube, microtiter well, sheet, bead,microparticle, chip, and other configurations known to those of ordinaryskill in the art.

“Specific binding” or “specifically binding” as used herein may refer tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, wherein the interaction is dependent upon the presenceof a particular structure (e.g., an antigenic determinant or epitope) onthe chemical species; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzymes and enzyme inhibitors, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes and fragments thereof, whether isolatedor recombinantly produced.

“Statistically significant” as used herein refers to the likelihood thata relationship between two or more variables is caused by somethingother than random chance. Statistical hypothesis testing is used todetermine whether the result of a data set is statistically significant.In statistical hypothesis testing, a statistical significant result isattained whenever the observed p-value of a test statistic is less thanthe significance level defined of the study. The p-value is theprobability of obtaining results at least as extreme as those observed,given that the null hypothesis is true. Examples of statisticalhypothesis analysis include Wilcoxon signed-rank test, t-test,Chi-Square or Fisher's exact test. “Significant” as used herein refersto a change that has not been determined to be statistically significant(e.g., it may not have been subject to statistical hypothesis testing).

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. In someembodiments, the subject is a human. The subject or patient may beundergoing other forms of treatment.

“Treat,” “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease and/or injury, or one or more symptoms of such disease, to whichsuch term applies. Depending on the condition of the subject, the termalso refers to preventing a disease, and includes preventing the onsetof a disease, or preventing the symptoms associated with a disease. Atreatment may be either performed in an acute or chronic way. The termalso refers to reducing the severity of a disease or symptoms associatedwith such disease prior to affliction with the disease. Such preventionor reduction of the severity of a disease prior to affliction refers toadministration of a pharmaceutical composition to a subject that is notat the time of administration afflicted with the disease. “Preventing”also refers to preventing the recurrence of a disease or of one or moresymptoms associated with such disease. “Treatment” and“therapeutically,” refer to the act of treating, as “treating” isdefined above.

“Traumatic Brain Injury” or “TBI” as used interchangeably herein refersto a complex injury with a broad spectrum of symptoms and disabilities.TBI is most often an acute event similar to other injuries. TBI can beclassified as “mild,” “moderate,” or “severe.” The causes of TBI arediverse and include, for example, physical shaking by a person, a caraccident, injuries from firearms, cerebral vascular accidents (e.g.,strokes), falls, explosions or blasts and other types of blunt forcetrauma. Other causes of TBI include the ingestion and/or exposure to oneor more chemicals or toxins (such as fires, molds, asbestos, pesticidesand insecticides, organic solvents, paints, glues, gases (such as carbonmonoxide, hydrogen sulfide, and cyanide), organic metals (such as methylmercury, tetraethyl lead and organic tin), one or more drugs of abuse orcombinations thereof). Alternatively, TBI can occur in subjectssuffering from an autoimmune disease, a metabolic disorder, a braintumor, hypoxia, one or more viruses, meningitis, hydrocephalus orcombinations thereof. Young adults and the elderly are the age groups athighest risk for TBI. In certain embodiments herein, traumatic braininjury or TBI does not include and specifically excludes cerebralvascular accidents such as strokes.

“Mild TBI” as used herein refers to a brain injury where loss ofconsciousness is brief and usually a few seconds or minutes and/orconfusion and disorientation is shorter than 1 hour. Mild TBI is alsoreferred to as a concussion, minor head trauma, minor TBI, minor braininjury, and minor head injury. While MRI and CT scans are often normal,the individual with mild TBI may have cognitive problems such asheadache, difficulty thinking, memory problems, attention deficits, moodswings and frustration.

Mild TBI is the most prevalent TBI and is often missed at time ofinitial injury. Typically, a subject has a Glasgow Coma scale number ofbetween 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of peoplewith mild TBI have symptoms that last 3 months or more. Mild TBI isdefined as the result of the forceful motion of the head or impactcausing a brief change in mental status (confusion, disorientation orloss of memory) or loss of consciousness for less than 30 minutes.Common symptoms of mild TBI include fatigue, headaches, visualdisturbances, memory loss, poor attention/concentration, sleepdisturbances, dizziness/loss of balance, irritability-emotionaldisturbances, feelings of depression, and seizures. Other symptomsassociated with mild TBI include nausea, loss of smell, sensitivity tolight and sounds, mood changes, getting lost or confused, and/orslowness in thinking.

“Moderate TBI” as used herein refers to a brain injury where loss ofconsciousness and/or confusion and disorientation is between 1 and 24hours and the subject has a Glasgow Coma scale number of between 9-13(such as 9-12 or 9-13). The individual with moderate TBI have abnormalbrain imaging results. “Severe TBI” as used herein refers to a braininjury where loss of consciousness is more than 24 hours and memory lossafter the injury or penetrating skull injury longer than 24 hours andthe subject has a Glasgow Coma scale number between 3-8. The deficitsrange from impairment of higher level cognitive functions to comatosestates. Survivors may have limited function of arms or legs, abnormalspeech or language, loss of thinking ability or emotional problems.Individuals with severe injuries can be left in long-term unresponsivestates. For many people with severe TBI, long-term rehabilitation isoften necessary to maximize function and independence.

“Moderate to severe” TBI as used herein refers to a spectrum of braininjury that includes moderate to severe and thus encompasses moderateTBI alone, severe TBI alone and moderate to severe TBI combined.Subjects suffering from a moderate to severe TBI can have a Glasgow Comascale number of between 3-13 (such as 3-12 or 3-13). For example, insome clinical situations, a subject may initially be diagnosed as havinga moderate TBI but who, over the course of time (minutes, hours ordays), progress to having a severe TBI (such, as for example, insituations when there is a brain bleed). Such subjects would be examplesof patients that could be classified as “moderate to severe”. Commonsymptoms of moderate to severe TBI include cognitive deficits includingdifficulties with attention, concentration, distractibility, memory,speed of processing, confusion, perseveration, impulsiveness, languageprocessing, and/or “executive functions”, not understanding the spokenword (receptive aphasia), difficulty speaking and being understood(expressive aphasia), slurred speech, speaking very fast or very slow,problems reading, problems writing, difficulties with interpretation oftouch, temperature, movement, limb position and fine discrimination, theintegration or patterning of sensory impressions into psychologicallymeaningful data, partial or total loss of vision, weakness of eyemuscles and double vision (diplopia), blurred vision, problems judgingdistance, involuntary eye movements (nystagmus), intolerance of light(photophobia), hearing, such as decrease or loss of hearing, ringing inthe ears (tinnitus), increased sensitivity to sounds, loss or diminishedsense of smell (anosmia), loss or diminished sense of taste, theconvulsions associated with epilepsy that can be several types and caninvolve disruption in consciousness, sensory perception, or motormovements, control of bowel and bladder, sleep disorders, loss ofstamina, appetite changes, regulation of body temperature, menstrualdifficulties, dependent behaviors, emotional ability, lack ofmotivation, irritability, aggression, depression, disinhibition, ordenial/lack of awareness.

“Ubiquitin carboxy-terminal hydrolase L1” or “UCH-L1” as usedinterchangeably herein refers to a deubiquitinating enzyme encoded bythe UCH-L1 gene in humans. UCH-L1, also known as ubiquitincarboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a memberof a gene family whose products hydrolyze small C-terminal adducts ofubiquitin to generate the ubiquitin monomer.

“UCH-L1 status” can mean either the level or amount of UCH-L1 at a pointin time (such as with a single measure of UCH-L1), the level or amountof UCH-L1 associated with monitoring (such as with a repeat test on asubject to identify an increase or decrease in UCH-L1 amount), the levelor amount of UCH-L1 associated with treatment for traumatic brain injury(whether a primary brain injury and/or a secondary brain injury) orcombinations thereof.

“Variant” is used herein to describe a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Representative examples of “biological activity”include the ability to be bound by a specific antibody or to promote animmune response. Variant is also used herein to describe a protein withan amino acid sequence that is substantially identical to a referencedprotein with an amino acid sequence that retains at least one biologicalactivity. A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity, degree, and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to refer to an antigenically reactivefragment of an anti-UCH-L1 antibody that differs from the correspondingfragment of anti-UCH-L1 antibody in amino acid sequence but is stillantigenically reactive and can compete with the corresponding fragmentof anti-UCH-L1 antibody for binding with UCH-L1. “Variant” also can beused to describe a polypeptide or a fragment thereof that has beendifferentially processed, such as by proteolysis, phosphorylation, orother post-translational modification, yet retains its antigenreactivity.

“Vector” is used herein to describe a nucleic acid molecule that cantransport another nucleic acid to which it has been linked. One type ofvector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors can replicate autonomously in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”). Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. “Plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions, can be used. In this regard,RNA versions of vectors (including RNA viral vectors) may also find usein the context of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. For example,any nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those that are well known and commonly used in the art. Themeaning and scope of the terms should be clear; in the event, however ofany latent ambiguity, definitions provided herein take precedent overany dictionary or extrinsic definition. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

2. Methods of Aiding in the Determination of Whether to Perform Imagingon a Human Subject Who has Sustained an Injury to the Head Using OneSample from the Subject

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform an imaging procedure, such asMRI or CT scan, on a human subject who has sustained or may havesustained an injury to the head. As used here, “determination of whetherto perform an imaging procedure, such as MRI or CT scan, on a humansubject” refers to the fact that the aforementioned method can be used,e.g., with other information (e.g., clinical assessment data), todetermine that the subject is more likely than not to have a positiveMRI scan or positive head CT scan, i.e., the presence of an intracraniallesion. Specifically, such a method can comprise the steps of: (a)performing an assay on a sample obtained from the subject within about24 hours after a suspected injury to the head to measure or detect alevel of an early biomarker in the sample, said early biomarkercomprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glialfibrillary acidic protein (GFAP), or a combination thereof, in thesample; and (b) performing an imaging procedure, such as MRI or CT scanon the subject when the level of the early biomarker in the sample ishigher than a reference level of the early biomarker (“rule in”performing an imaging procedure) and not performing an imaging procedureon the subject when the level of the early biomarker in the sample islower than a reference level of the early biomarker (“rule out”performing an imaging procedure). The sample can be a biological sample.

In some embodiments, the method can include obtaining a sample withinabout 24 hours of a suspected injury to the subject and contacting thesample with an antibody for an early biomarker of TBI, such as ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof, to allow formation of a complex of theantibody and the early biomarker. The method also includes detecting theresulting antibody-early biomarker complex.

In some embodiments, the sample is taken from the human subject withinabout 24 hours of injury or suspected injury to the head, such as withinabout 0 to about 6 hours, within about 0 to about 8 hours, within about0 to about 10 hours, within about 0 to about 12 hours, within about 0 toabout 18 hours, within about 6 hours to about 12 hours, within about 6hours to about 18 hours, or within about 12 hours to about 18 hours. Forexample, the sample can be taken from the human subject within about 0minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, 7hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or about 24 hours ofinjury or suspected injury to the head. In some embodiments, the onsetof the presence of the early biomarker, such as UCH-L1, GFAP, or acombination thereof, appears within about 0 minutes, about 30 minutes,about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours,about 4 hours, about 5 hours, about 6 hours, 7 hours, about 8 hours,about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours,about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22hours, about 23 hours, or about 24 hours after injury to the head.

In some embodiments, the subject has received an imaging procedure, suchas MRI or CT scan, before or after the assay is performed. In someembodiments, the subject is suspected as having a traumatic brain injurybased on the imaging procedure. In some embodiments, the reference levelof the early biomarker, such as UCH-L1, GFAP, or a combination thereof,is correlated with a positive MRI scan or positive head CT scan, i.e.,the presence of an intracranial lesion.

Generally, a reference level of the early biomarker, such as UCH-L1,GFAP, or a combination thereof, can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for UCH-L1.Generally, in making such a comparison, the reference level of the earlybiomarker, such as UCH-L1, GFAP, or a combination thereof, is obtainedby running a particular assay a sufficient number of times and underappropriate conditions such that a linkage or association of analytepresence, amount or concentration with a particular stage or endpoint ofTBI or with particular indicia can be made. Typically, the referencelevel of the early biomarker, such as UCH-L1, GFAP, or a combinationthereof, is obtained with assays of reference subjects (or populationsof subjects). The early biomarker, such as UCH-L1, GFAP, or acombination thereof, measured can include fragments thereof, degradationproducts thereof, and/or enzymatic cleavage products thereof.

In some embodiments, the reference level of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, is determined by an assay havinga sensitivity of between at least about 70% to about 100% and aspecificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 70% to about100%, between at least about 70% to at least about 99%, between at leastabout 70% to at least about 95%, between at least about 70% to at leastabout 90%, between at least about 70% to at least about 85%, between atleast about 75% to about 100%, between at least about 75% to at leastabout 99%, between at least about 75% to at least about 95%, between atleast about 75% to at least about 90%, between at least about 75% to atleast about 85%, between at least about 80% to about 100%, between atleast about 80% to at least about 99%, between at least about 80% to atleast about 95%, between at least about 80% to at least about 90%,between at least about 80% to at least about 85%, between at least about85% to about 100%, between at least about 85% to at least about 99%,between at least about 85% to at least about 95%, between at least about85% to at least about 90%, between at least about 90% to about 100%,between at least about 90% to at least about 99%, between at least about90% to at least about 95%, between at least about 95% to about 100%, orbetween at least about 95% to at least about 99%. In some embodiments,the sensitivity is at least about 70.0%, at least about 75.0%, at leastabout 80.0%, at least about 85.0%, at least about 87.5%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the reference level of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, can be between at least about 10pg/mL to about 500 pg/mL. In some embodiments, the reference level ofthe early biomarker, such as UCH-L1, GFAP, or a combination thereof, canbe between at least about 10 pg/mL to about 500 pg/mL, between at leastabout 10 pg/mL to about 400 pg/mL, between at least about 10 pg/mL toabout 300 pg/mL, between at least about 10 pg/mL to about 200 pg/mL,between at least about 10 pg/mL to about 100 pg/mL, between at leastabout 10 pg/mL to about 50 pg/mL, between at least about 10 pg/mL toabout 40 pg/mL, between at least about 10 pg/mL to about 30 pg/mL,between at least about 20 pg/mL to about 500 pg/mL, between at leastabout 20 pg/mL to about 400 pg/mL, between at least about 20 pg/mL toabout 300 pg/mL, between at least about 20 pg/mL to about 200 pg/mL,between at least about 20 pg/mL to about 100 pg/mL, between at leastabout 20 pg/mL to about 50 pg/mL, between at least about 20 pg/mL toabout 40 pg/mL, between at least about 20 pg/mL to about 30 pg/mL,between at least about 30 pg/mL to about 500 pg/mL, between at leastabout 30 pg/mL to about 400 pg/mL, between at least about 30 pg/mL toabout 300 pg/mL, between at least about 30 pg/mL to about 200 pg/mL,between at least about 30 pg/mL to about 100 pg/mL, between at leastabout 30 pg/mL to about 50 pg/mL, between at least about 30 pg/mL toabout 40 pg/mL, between at least about 40 pg/mL to about 500 pg/mL,between at least about 40 pg/mL to about 400 pg/mL, between at leastabout 40 pg/mL to about 300 pg/mL, between at least about 40 pg/mL toabout 200 pg/mL, between at least about 40 pg/mL to about 100 pg/mL,between at least about 40 pg/mL to about 50 pg/mL, between at leastabout 50 pg/mL to about 500 pg/mL, between at least about 50 pg/mL toabout 400 pg/mL, between at least about 50 pg/mL to about 300 pg/mL,between at least about 50 pg/mL to about 200 pg/mL, between at leastabout 50 pg/mL to about 100 pg/mL, between at least about 75 pg/mL toabout 500 pg/mL, between at least about 75 pg/mL to about 400 pg/mL,between at least about 75 pg/mL to about 300 pg/mL, between at leastabout 75 pg/mL to about 200 pg/mL, between at least about 75 pg/mL toabout 100 pg/mL, between at least about 100 pg/mL to about 500 pg/mL,between at least about 100 pg/mL to about 400 pg/mL, between at leastabout 100 pg/mL to about 300 pg/mL, between at least about 100 pg/mL toabout 200 pg/mL, between at least about 150 pg/mL to about 500 pg/mL,between at least about 150 pg/mL to about 400 pg/mL, between at leastabout 150 pg/mL to about 300 pg/mL, between at least about 150 pg/mL toabout 200 pg/mL, between at least about 200 pg/mL to about 500 pg/mL,between at least about 200 pg/mL to about 400 pg/mL, or between at leastabout 200 pg/mL to about 300 pg/mL. For example, the reference level forUCH-L1 can be between at least about 80 pg/mL to about 150 pg/mL and thereference level for GFAP can be between at least about 20 pg/mL to about200 pg/mL.

In some embodiments, the reference level of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, can be at least about 10 pg/mL,at least about 20 pg/mL, at least about 30 pg/mL, at least about 40pg/mL, at least about 50 pg/mL, at least about 55 pg/mL, at least about60 pg/mL, at least about 65 pg/mL, at least about 70 pg/mL, at leastabout 75 pg/mL, at least about 80 pg/mL, at least about 85 pg/mL, atleast about 90 pg/mL, at least about 95 pg/mL, at least about 100 pg/mL,at least about 150 pg/mL, at least about 200 pg/mL, at least about 250pg/mL, at least about 300 pg/mL, at least about 350 pg/mL, at leastabout 400 pg/mL, at least about 450 pg/mL, or at least about 500 pg/mL.In some embodiments, the reference level for GFAP is between about 20pg/mL and about 200 pg/mL. In some embodiments, the reference level forUCH-L1 is about 80 pg/mL and about 150 pg/mL.

In some embodiments, the method further includes treating the humansubject with a traumatic brain injury treatment and/or monitoring thehuman subject, as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS). Also, the assay can be employed in a clinical chemistry formatsuch as would be known by one of ordinary skill in the art. Such assaysare described in further detail herein in Sections 5-9. It is known inthe art that the values (e.g., reference levels, cutoffs, thresholds,specificities, sensitivities, concentrations of calibrators and/orcontrols etc.) used in an assay that employs specific sample type (e.g.,such as an immunoassay that utilizes serum or a point-of-care devicethat employs whole blood) can be extrapolated to other assay formatsusing known techniques in the art, such as assay standardization. Forexample, one way in which assay standardization can be performed is byapplying a factor to the calibrator employed in the assay to make thesample concentration read higher or lower to get a slope that alignswith the comparator method. Other methods of standardizing resultsobtained on one assay to another assay are well known and have beendescribed in the literature (See, for example, David Wild, ImmunoassayHandbook, 4^(th) edition, chapter 3.5, pages 315-322, the contents ofwhich are herein incorporated by reference).

3. Methods of Aiding in the Determination of Whether to Perform Imagingon a Human Subject Who has Sustained an Injury to the Head Using atLeast Two Samples from the Subject

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform an imaging procedure, such asMRI or CT scan, on a human subject who has sustained or may havesustained an injury to the head. As used here, “determination of whetherto perform an imaging procedure, such as MRI or CT scan, on a humansubject” refers to the fact that the aforementioned method can be used,e.g., with other information (e.g., clinical assessment data), todetermine that the subject is more likely than not to have a positiveMRI scan or positive head CT scan, i.e., the presence of an intracraniallesion. Specifically, such a method can comprise the steps of:performing an assay on at least two samples obtained from the subject,the first sample taken from the subject within about 24 hours of asuspected injury and the second sample taken from the subject from about3 to about 6 hours after the first sample is taken; detecting in the atleast two samples an early biomarker of traumatic brain injury, such asubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillaryacidic protein (GFAP), or a combination thereof; and performing animaging procedure, such as MRI or CT scan, on the subject when the levelof the early biomarker decreases or increases by at least an absoluteamount from the first sample to the second sample (“rule in” performingan imaging procedure) and not performing an imaging procedure on thesubject when there is no decrease or increase by at least an absoluteamount in the level of the early biomarker from the first sample to thesecond sample (“rule out” performing an imaging procedure). The samplescan be biological samples.

In some embodiments, the method can include contacting the samples withan antibody for an early biomarker of TBI, such as ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof, to allow formation of a complex of theantibody and the early biomarker. The method also includes detecting theresulting antibody-early biomarker complex to determine the levels ofthe early biomarker, such as UCH-L1, GFAP, or a combination thereof, foreach of the first sample and second sample. The onset of the presence ofthe early biomarker, such as UCH-L1, GFAP, or a combination thereof,appears within about 0 to about 24 hours after the onset of thesuspected injury. In some embodiments, the onset of the presence of theearly biomarker, such as UCH-L1, GFAP, or a combination thereof, appearswithin about 0 minutes, about 30 minutes, about 60 minutes, about 90minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours,about 6 hours, 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about24 hours after injury to the head.

In some embodiments, a first sample is obtained at a first time pointwithin about 24 hours of the suspected injury and a second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point to determine whether thesubject will have a positive or negative MRI scan or head CT scan, i.e.,the presence or absence of an intracranial lesion. In some embodiments,the first sample is taken within about 24 hours after the suspectedinjury and the second sample is taken within about 3 hours to about 6hours after the first sample. In some embodiments, the first time pointis about 0 to about 24 hours after the injury or suspected injury to thehead. For example, the first time point can be within about 0 to about 6hours, within about 0 to about 8 hours, within about 0 to about 10hours, within about 0 to about 12 hours, within about 0 to about 18hours, within about 6 hours to about 12 hours, within about 6 hours toabout 18 hours, or within about 12 hours to about 18 hours after theinjury or suspected injury to the head. For example, the first samplecan be taken from the human subject within about 0 minutes, about 30minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3hours, about 4 hours, about 5 hours, about 6 hours, 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours of injury or suspectedinjury to the head after the suspected injury.

In some embodiments, the second time point, or optionally a third timepoint or fourth time point, is about 1 hour to about 10 hours after thefirst time point, such as about 3 hours to about 6 hours after the firsttime point. In some embodiments, the second time point is about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hoursafter the first time point.

In some embodiments, the reference level of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, is determined by an assay havinga sensitivity of between at least about 70% to about 100% and aspecificity of between at least about 30% to about 100%. In someembodiments, the sensitivity is between at least about 70% to about100%, between at least about 70% to at least about 99%, between at leastabout 70% to at least about 95%, between at least about 70% to at leastabout 90%, between at least about 70% to at least about 85%, between atleast about 75% to about 100%, between at least about 75% to at leastabout 99%, between at least about 75% to at least about 95%, between atleast about 75% to at least about 90%, between at least about 75% to atleast about 85%, between at least about 80% to about 100%, between atleast about 80% to at least about 99%, between at least about 80% to atleast about 95%, between at least about 80% to at least about 90%,between at least about 80% to at least about 85%, between at least about85% to about 100%, between at least about 85% to at least about 99%,between at least about 85% to at least about 95%, between at least about85% to at least about 90%, between at least about 90% to about 100%,between at least about 90% to at least about 99%, between at least about90% to at least about 95%, between at least about 95% to about 100%, orbetween at least about 95% to at least about 99%. In some embodiments,the sensitivity is at least about 70.0%, at least about 75.0%, at leastabout 80.0%, at least about 85.0%, at least about 87.5%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the absolute amount can be between at least about 5pg/mL to about 1000 pg/mL. In some embodiments, the absolute amount canbe between at least about 5 pg/mL to about 1000 pg/mL, between at leastabout 5 pg/mL to about 750 pg/mL, between at least about 5 pg/mL toabout 500 pg/mL, between at least about 5 pg/mL to about 400 pg/mL,between at least about 5 pg/mL to about 300 pg/mL, between at leastabout 5 pg/mL to about 200 pg/mL, between at least about 5 pg/mL toabout 100 pg/mL, between at least about 5 pg/mL to about 50 pg/mL,between at least about 10 pg/mL to about 1000 pg/mL, between at leastabout 10 pg/mL to about 750 pg/mL, between at least about 10 pg/mL toabout 500 pg/mL, between at least about 10 pg/mL to about 400 pg/mL,between at least about 10 pg/mL to about 300 pg/mL, between at leastabout 10 pg/mL to about 200 pg/mL, between at least about 10 pg/mL toabout 100 pg/mL, between at least about 10 pg/mL to about 50 pg/mL,between at least about 20 pg/mL to about 1000 pg/mL, between at leastabout 20 pg/mL to about 750 pg/mL, between at least about 20 pg/mL toabout 500 pg/mL, between at least about 20 pg/mL to about 400 pg/mL,between at least about 20 pg/mL to about 300 pg/mL, between at leastabout 20 pg/mL to about 200 pg/mL, between at least about 20 pg/mL toabout 100 pg/mL, between at least about 20 pg/mL to about 50 pg/mL,between at least about 25 pg/mL to about 1000 pg/mL, between at leastabout 25 pg/mL to about 750 pg/mL, between at least about 25 pg/mL toabout 500 pg/mL, between at least about 25 pg/mL to about 400 pg/mL,between at least about 25 pg/mL to about 300 pg/mL, between at leastabout 25 pg/mL to about 200 pg/mL, between at least about 25 pg/mL toabout 100 pg/mL, between at least about 25 pg/mL to about 50 pg/mL,between at least about 50 pg/mL to about 1000 pg/mL, between at leastabout 50 pg/mL to about 750 pg/mL, between at least about 50 pg/mL toabout 500 pg/mL, between at least about 50 pg/mL to about 400 pg/mL,between at least about 50 pg/mL to about 300 pg/mL, between at leastabout 50 pg/mL to about 200 pg/mL, between at least about 50 pg/mL toabout 100 pg/mL, between at least about 100 pg/mL to about 1000 pg/mL,between at least about 100 pg/mL to about 750 pg/mL, between at leastabout 100 pg/mL to about 500 pg/mL, between at least about 100 pg/mL toabout 400 pg/mL, between at least about 100 pg/mL to about 300 pg/mL,between at least about 100 pg/mL to about 200 pg/mL, between at leastabout 200 pg/mL to about 1000 pg/mL, between at least about 200 pg/mL toabout 750 pg/mL, between at least about 200 pg/mL to about 500 pg/mL,between at least about 200 pg/mL to about 400 pg/mL, between at leastabout 200 pg/mL to about 300 pg/mL, between at least about 300 pg/mL toabout 1000 pg/mL, between at least about 300 pg/mL to about 750 pg/mL,between at least about 300 pg/mL to about 500 pg/mL, between at leastabout 300 pg/mL to about 400 pg/mL, between at least about 400 pg/mL toabout 1000 pg/mL, between at least about 400 pg/mL to about 750 pg/mL,or between at least about 400 pg/mL to about 500 pg/mL. In someembodiments, the absolute amount can be at least about 5 pg/mL, at leastabout 6 pg/mL, at least about 7 pg/mL, at least about 8 pg/mL, at leastabout 9 pg/mL, at least about 10 pg/mL, at least about 11 pg/mL, atleast about 12 pg/mL, at least about 13 pg/mL, at least about 14 pg/mL,at least about 15 pg/mL, at least about 16 pg/mL, at least about 17pg/mL, at least about 18 pg/mL, at least about 19 pg/mL, at least about20 pg/mL, at least about 21 pg/mL, at least about 22 pg/mL, at leastabout 23 pg/mL, at least about 24 pg/mL, at least about 25 pg/mL, atleast about 26 pg/mL, at least about 27 pg/mL, at least about 28 pg/mL,at least about 29 pg/mL, at least about 30 pg/mL, at least about 35pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, at least about50 pg/mL, at least about 55 pg/mL, at least about 60 pg/mL, at leastabout 65 pg/mL, at least about 70 pg/mL, at least about 75 pg/mL, atleast about 80 pg/mL, at least about 85 pg/mL, at least about 90 pg/mL,at least about 95 pg/mL, at least about 100 pg/mL, at least about 110pg/mL, at least about 120 pg/mL, at least about 129 pg/mL, at leastabout 130 pg/mL, at least about 140 pg/mL, at least about 150 pg/mL, atleast about 200 pg/mL, at least about 250 pg/mL, at least about 300pg/mL, at least about 350 pg/mL, at least about 400 pg/mL, at leastabout 450 pg/mL, at least about 500 pg/mL, at least about 550 pg/mL, atleast about 600 pg/mL, at least about 650 pg/mL, at least about 700pg/mL, at least about 750 pg/mL, at least about 800 pg/mL, at leastabout 900 pg/mL, or at least about 1000 pg/mL.

In some embodiments, the early biomarker is UCH-L1 and the absoluteamount is between at least about 30 pg/mL to about 100 pg/mL, the earlybiomarker is GFAP and the absolute amount is between at least about 10pg/mL to about 150 pg/mL, or a combination thereof.

In some embodiments, the method further includes treating the humansubject with a traumatic brain injury treatment and/or monitoring thehuman subject, as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS). Also, the assay can be employed in a clinical chemistry formatsuch as would be known by one of ordinary skill in the art. Such assaysare described in further detail herein in Sections 5-9. It is known inthe art that the values (e.g., reference levels, cutoffs, thresholds,specificities, sensitivities, concentrations of calibrators and/orcontrols etc.) used in an assay that employs specific sample type (e.g.,such as an immunoassay that utilizes serum or a point-of-care devicethat employs whole blood) can be extrapolated to other assay formatsusing known techniques in the art, such as assay standardization. Forexample, one way in which assay standardization can be performed is byapplying a factor to the calibrator employed in the assay to make thesample concentration read higher or lower to get a slope that alignswith the comparator method. Other methods of standardizing resultsobtained on one assay to another assay are well known and have beendescribed in the literature (See, for example, David Wild, ImmunoassayHandbook, 4^(th) edition, chapter 3.5, pages 315-322, the contents ofwhich are herein incorporated by reference).

4. Treatment and Monitoring of Subjects Who have Sustained an Injury tothe Head

The subject identified in the methods described above may be treated ormonitored. In some embodiments, the method further includes treating thehuman subject with a traumatic brain injury treatment, such as anytreatments known in the art. For example, treatment of traumatic braininjury can take a variety of forms depending on the severity of theinjury to the head. For example, for subjects suffering from mild TBI,the treatment may include one or more of rest, abstaining from physicalactivities, such as sports, avoiding light or wearing sunglasses whenout in the light, medication for relief of a headache or migraine,anti-nausea medication, etc. Treatment for patients suffering frommoderate, severe or moderate to severe TBI might include administrationof one or more appropriate medications (such as, for example, diuretics,anti-convulsant medications, medications to sedate and put an individualin a drug-induced coma, or other pharmaceutical or biopharmaceuticalmedications (either known or developed in the future for treatment ofTBI), one or more surgical procedures (such as, for example, removal ofa hematoma, repairing a skull fracture, decompressive craniectomy,etc.), protecting the airway, and one or more therapies (such as, forexample one or more rehabilitation, cognitive behavioral therapy, angermanagement, counseling psychology, etc.). In some embodiments, themethod further includes monitoring the human subject. In someembodiments, a subject may be monitored with CT scan or MRI.

5. Methods for Measuring the Level of UCH-L1

In the methods described above, UCH-L1 levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of UCH-L1 includes contactingthe sample with a first specific binding member and second specificbinding member. In some embodiments the first specific binding member isa capture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of UCH-L1 includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., UCH-L1-capture antibody), whichbinds to an epitope on UCH-L1 or UCH-L1 fragment to form a captureantibody-UCH-L1 antigen complex (e.g., UCH-L1-capture antibody-UCH-L1antigen complex), and (2) a detection antibody (e.g., UCH-L1-detectionantibody), which includes a detectable label and binds to an epitope onUCH-L1 that is not bound by the capture antibody, to form a UCH-L1antigen-detection antibody complex (e.g., UCH-L1antigen-UCH-L1-detection antibody complex), such that a captureantibody-UCH-L1 antigen-detection antibody complex (e.g., UCH-L1-captureantibody-UCH-L1 antigen-UCH-L1-detection antibody complex) is formed,and measuring the amount or concentration of UCH-L1 in the sample basedon the signal generated by the detectable label in the captureantibody-UCH-L1 antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a UCH-L1 antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example the Abbott Laboratoriesinstrument ARCHITECT®, and other core laboratory instruments) other thana point-of-care device may be capable of measuring levels of UCH-L1 in asample higher or greater than 25,000 pg/mL.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety

6. UCH-L1 Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to ubiquitin carboxy-terminal hydrolase L1 (“UCH-L1”)(or fragments thereof), referred to as “UCH-L1 antibody.” The UCH-L1antibodies can be used to assess the UCH-L1 status as a measure oftraumatic brain injury, detect the presence of UCH-L1 in a sample,quantify the amount of UCH-L1 present in a sample, or detect thepresence of and quantify the amount of UCH-L1 in a sample.

a. Ubiquitin Carboxy-Terminal Hydrolase L1 (UCH-L1)

Ubiquitin carboxy-terminal hydrolase L1 (“UCH-L1”), which is also knownas “ubiquitin C-terminal hydrolase,” is a deubiquitinating enzyme.UCH-L1 is a member of a gene family whose products hydrolyze smallC-terminal adducts of ubiquitin to generate the ubiquitin monomer.Expression of UCH-L1 is highly specific to neurons and to cells of thediffuse neuroendocrine system and their tumors. It is abundantly presentin all neurons (accounts for 1-2% of total brain protein), expressedspecifically in neurons and testis/ovary. The catalytic triad of UCH-L1contains a cysteine at position 90, an aspartate at position 176, and ahistidine at position 161 that are responsible for its hydrolaseactivity.

Human UCH-L1 may have the following amino acid sequence:

(SEQ ID NO: 1) MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA.

The human UCH-L1 may be a fragment or variant of SEQ ID NO: 1. Thefragment of UCH-L1 may be between 5 and 225 amino acids, between 10 and225 amino acids, between 50 and 225 amino acids, between 60 and 225amino acids, between 65 and 225 amino acids, between 100 and 225 aminoacids, between 150 and 225 amino acids, between 100 and 175 amino acids,or between 175 and 225 amino acids in length. The fragment may comprisea contiguous number of amino acids from SEQ ID NO: 1.

b. UCH-L1-Recognizing Antibody

The antibody is an antibody that binds to UCH-L1, a fragment thereof, anepitope of UCH-L1, or a variant thereof. The antibody may be a fragmentof the anti-UCH-L1 antibody or a variant or a derivative thereof. Theantibody may be a polyclonal or monoclonal antibody. The antibody may bea chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, a fully human antibodyor an antibody fragment, such as a Fab fragment, or a mixture thereof.Antibody fragments or derivatives may comprise F(ab′)₂, Fv or scFvfragments. The antibody derivatives can be produced by peptidomimetics.Further, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies.

The anti-UCH-L1 antibodies may be a chimeric anti-UCH-L1 or humanizedanti-UCH-L1 antibody. In one embodiment, both the humanized antibody andchimeric antibody are monovalent. In one embodiment, both the humanizedantibody and chimeric antibody comprise a single Fab region linked to anFc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-UCH-L1 antibodies. For example, librariesof naïve human single chain variable fragments (scFv) may be used toselect human anti-UCH-L1 antibodies. Transgenic animals may be used toexpress human antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

(1) Epitope

The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 1), afragment thereof, or a variant thereof. The antibody mayimmunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

c. Antibody Preparation/Production

Antibodies may be prepared by any of a variety of techniques, includingthose well known to those skilled in the art. In general, antibodies canbe produced by cell culture techniques, including the generation ofmonoclonal antibodies via conventional techniques, or via transfectionof antibody genes, heavy chains, and/or light chains into suitablebacterial or mammalian cell hosts, in order to allow for the productionof antibodies, wherein the antibodies may be recombinant. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies in either prokaryoticor eukaryotic host cells, expression of antibodies in eukaryotic cellsis preferable, and most preferable in mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesinclude Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody. Recombinant DNA technology may also be used to remove some, orall, of the DNA encoding either or both of the light and heavy chainsthat is not necessary for binding to the antigens of interest. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies. In addition, bifunctional antibodies maybe produced in which one heavy and one light chain are an antibody(i.e., binds human UCH-L1) and the other heavy and light chain arespecific for an antigen other than human UCH-L1 by crosslinking anantibody to a second antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the method of synthesizing a recombinant antibodymay be by culturing a host cell in a suitable culture medium until arecombinant antibody is synthesized. The method can further compriseisolating the recombinant antibody from the culture medium.

Methods of preparing monoclonal antibodies involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Such cell lines may be produced from spleen cells obtainedfrom an immunized animal. The animal may be immunized with UCH-L1 or afragment and/or variant thereof. The peptide used to immunize the animalmay comprise amino acids encoding human Fc, for example the fragmentcrystallizable region or tail region of human antibody. The spleen cellsmay then be immortalized by, for example, fusion with a myeloma cellfusion partner. A variety of fusion techniques may be employed. Forexample, the spleen cells and myeloma cells may be combined with anonionic detergent for a few minutes and then plated at low density on aselective medium that supports that growth of hybrid cells, but notmyeloma cells. One such technique uses hypoxanthine, aminopterin,thymidine (HAT) selection. Another technique includes electrofusion.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity may be used.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. Affinity chromatography is an example ofa method that can be used in a process to purify the antibodies.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment, which comprises bothantigen-binding sites.

The Fv fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecules. TheFv fragment may be derived using recombinant techniques. The Fv fragmentincludes a non-covalent VH::VL heterodimer including an antigen-bindingsite that retains much of the antigen recognition and bindingcapabilities of the native antibody molecule.

The antibody, antibody fragment, or derivative may comprise a heavychain and a light chain complementarity determining region (“CDR”) set,respectively interposed between a heavy chain and a light chainframework (“FR”) set which provide support to the CDRs and define thespatial relationship of the CDRs relative to each other. The CDR set maycontain three hypervariable regions of a heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g.,as available from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al.(1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J Immunol.17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.).; Gray etal. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134 (1994)).

An affinity matured antibody may be produced by any one of a number ofprocedures that are known in the art. For example, see Marks et al.,BioTechnology, 10: 779-783 (1992) describes affinity maturation by VHand VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

Antibody variants can also be prepared using delivering a polynucleotideencoding an antibody to a suitable host such as to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. These methods are known inthe art and are described for example in U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody variants also can be prepared by delivering a polynucleotide toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol.Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies can also beproduced using transgenic plants, according to known methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally, part or all of the non-humanor human CDR sequences are maintained while the non-human sequences ofthe variable and constant regions are replaced with human or other aminoacids.

Small antibody fragments may be diabodies having two antigen-bindingsites, wherein fragments comprise a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (VH VL). See for example, EP 404,097; WO 93/11161; and Hollingeret al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. See also,U.S. Pat. No. 6,632,926 to Chen et al. which is hereby incorporated byreference in its entirety and discloses antibody variants that have oneor more amino acids inserted into a hypervariable region of the parentantibody and a binding affinity for a target antigen which is at leastabout two fold stronger than the binding affinity of the parent antibodyfor the antigen.

The antibody may be a linear antibody. The procedure for making a linearantibody is known in the art and described in Zapata et al., (1995)Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pairof tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The antibodies may be recovered and purified from recombinant cellcultures by known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

It may be useful to detectably label the antibody. Methods forconjugating antibodies to these agents are known in the art. For thepurpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.They can be linked to a cytokine, to a ligand, to another antibody.Suitable agents for coupling to antibodies to achieve an anti-tumoreffect include cytokines, such as interleukin 2 (IL-2) and TumorNecrosis Factor (TNF); photosensitizers, for use in photodynamictherapy, including aluminum (III) phthalocyanine tetrasulfonate,hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131(131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re);antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinesecobra (naja naja atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestrictocin (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; ly207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

Antibody production via the use of hybridoma technology, the selectedlymphocyte antibody method (SLAM), transgenic animals, and recombinantantibody libraries is described in more detail below.

(1) Anti-UCH-L1 Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is alsonoted that the term “monoclonal antibody” as used herein is not limitedto antibodies produced through hybridoma technology. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

Methods of generating monoclonal antibodies as well as antibodiesproduced by the method may comprise culturing a hybridoma cell secretingan antibody of the invention wherein, preferably, the hybridoma isgenerated by fusing splenocytes isolated from an animal, e.g., a rat ora mouse, immunized with UCH-L1 with myeloma cells and then screening thehybridomas resulting from the fusion for hybridoma clones that secretean antibody able to bind a polypeptide of the invention. Briefly, ratscan be immunized with a UCH-L1 antigen. In a preferred embodiment, theUCH-L1 antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks; however, a single administration of thepolypeptide may also be used.

After immunization of an animal with a UCH-L1 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-UCH-L1antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-UCH-L1 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen UCH-L1 are detected in the rat serum, the rat spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example, cellsfrom cell line SP20 available from the American Type Culture Collection(ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding UCH-L1. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing rats with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using UCH-L1, or a portionthereof, or a cell expressing UCH-L1. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunosorbentassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. Anexample of ELISA screening is provided in PCT Publication No. WO00/37504.

Anti-UCH-L1 antibody-producing hybridomas are selected, cloned, andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production, and desirable antibodycharacteristics. Hybridomas may be cultured and expanded in vivo insyngeneic animals, in animals that lack an immune system, e.g., nudemice, or in cell culture in vitro. Methods of selecting, cloning andexpanding hybridomas are well known to those of ordinary skill in theart.

In a preferred embodiment, hybridomas are rat hybridomas. In anotherembodiment, hybridomas are produced in a non-human, non-rat species suchas mice, sheep, pigs, goats, cattle, or horses. In yet another preferredembodiment, the hybridomas are human hybridomas, in which a humannon-secretory myeloma is fused with a human cell expressing ananti-UCH-L1 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce two identical Fabfragments) or pepsin (to produce an F(ab′)₂ fragment). A F(ab′)₂fragment of an IgG molecule retains the two antigen-binding sites of thelarger (“parent”) IgG molecule, including both light chains (containingthe variable light chain and constant light chain regions), the CH1domains of the heavy chains, and a disulfide-forming hinge region of theparent IgG molecule. Accordingly, an F(ab′)₂ fragment is still capableof crosslinking antigen molecules like the parent IgG molecule.

(2) Anti-UCH-L1 Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook etal., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals are screened using anantigen-specific hemolytic plaque assay, wherein the antigen UCH-L1, asubunit of UCH-L1, or a fragment thereof, is coupled to sheep red bloodcells using a linker, such as biotin, and used to identify single cellsthat secrete antibodies with specificity for UCH-L1. Followingidentification of antibody-secreting cells of interest, heavy- andlight-chain variable region cDNAs are rescued from the cells by reversetranscriptase-PCR (RT-PCR) and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example, by panning thetransfected cells to isolate cells expressing antibodies to UCH-L1. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation method. See, for example, PCTPublication No. WO 97/29131 and PCT Publication No. WO 00/56772.

(3) Anti-UCH-L1 Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the invention, antibodies are produced byimmunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with a UCH-L1 antigen. In an embodiment, thenon-human animal is a transgenic mouse (XENOMOUSE®), an engineered mousestrain that comprises large fragments of the human immunoglobulin lociand is deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771;5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and6,130,364. See also PCT Publication Nos. WO 91/10741; WO 94/02602; WO96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human monoclonal antibodies.The XENOMOUSE® transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics, 15: 146-156 (1997),Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosuresof which are hereby incorporated by reference.

(4) Anti-UCH-L1 Monoclonal Antibodies Using Recombinant AntibodyLibraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired UCH-L1-binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, U.S. Pat. No. 5,223,409(Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCTPublication No. WO 91/17271 (Dower et al.); PCT Publication No. WO92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland etal.); PCT Publication No. WO 93/01288 (Breitling et al.); PCTPublication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO92/09690 (Garrard et al.); Fuchs et al., Bio/Technology, 9: 1369-1372(1991); Hay et al., Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse etal., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkinset al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature,352: 624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio/Technology, 9: 1373-1377 (1991);Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas etal., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); U.S. PatentApplication Publication No. 2003/0186374; and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

The recombinant antibody library may be from a subject immunized withUCH-L1, or a portion of UCH-L1. Alternatively, the recombinant antibodylibrary may be from a naive subject, i.e., one who has not beenimmunized with UCH-L1, such as a human antibody library from a humansubject who has not been immunized with human UCH-L1. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human UCH-L1 to thereby select thoseantibodies that recognize UCH-L1. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for UCH-L1, such as thosethat dissociate from human UCH-L1 with a particular K_(off) rateconstant, the art-known method of surface plasmon resonance can be usedto select antibodies having the desired K_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hUCH-L1, such as those with a particular IC₅₀, standard methodsknown in the art for assessing the inhibition of UCH-L1 activity may beused.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human UCH-L1. Preferably,the antibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. Such phage can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Phageexpressing an antigen binding domain that binds the antigen of interestcan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv, or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies include those disclosed in Brinkmannet al., J Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al.,Advances in Immunology, 57: 191-280 (1994); PCT Publication No. WO92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743; and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J Reprod. Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al.,Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al.,Science, 240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the invention. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98/31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology is PROfusion display technology.

In another approach, the antibodies can also be generated using yeastdisplay methods known in the art. In yeast display methods, geneticmethods are used to tether antibody domains to the yeast cell wall anddisplay them on the surface of yeast. In particular, such yeast can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Examples ofyeast display methods that can be used to make the antibodies includethose disclosed in U.S. Pat. No. 6,699,658 (Wittrup et al.) incorporatedherein by reference.

d. Production of Recombinant UCH-L1 Antibodies

Antibodies may be produced by any of a number of techniques known in theart. For example, expression from host cells, wherein expressionvector(s) encoding the heavy and light chains is (are) transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection, and the like. Although it ispossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody of this invention. Recombinant DNA technology may also be usedto remove some, or all, of the DNA encoding either or both of the lightand heavy chains that is not necessary for binding to the antigens ofinterest. The molecules expressed from such truncated DNA molecules arealso encompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention (i.e., binds human UCH-L1) andthe other heavy and light chain are specific for an antigen other thanhuman UCH-L1 by crosslinking an antibody of the invention to a secondantibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

(1) Humanized Antibody

The humanized antibody may be an antibody or a variant, derivative,analog or portion thereof which immunospecifically binds to an antigenof interest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. The humanized antibody may befrom a non-human species antibody that binds the desired antigen havingone or more complementarity determining regions (CDRs) from thenon-human species and framework regions from a human immunoglobulinmolecule.

As used herein, the term “substantially” in the context of a CDR refersto a CDR having an amino acid sequence at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′,F(ab′)₂, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. According to one aspect, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or of a heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3, and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In one embodiment, suchmutations, however, will not be extensive. Usually, at least 90%, atleast 95%, at least 98%, or at least 99% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences.As used herein, the term “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence. As used herein, theterm “consensus immunoglobulin sequence” refers to the sequence formedfrom the most frequently occurring amino acids (or nucleotides) in afamily of related immunoglobulin sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In afamily of immunoglobulins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

The humanized antibody may be designed to minimize unwantedimmunological response toward rodent anti-human antibodies, which limitsthe duration and effectiveness of therapeutic applications of thosemoieties in human recipients. The humanized antibody may have one ormore amino acid residues introduced into it from a source that isnon-human. These non-human residues are often referred to as “import”residues, which are typically taken from a variable domain. Humanizationmay be performed by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. For example, see U.S.Pat. No. 4,816,567, the contents of which are herein incorporated byreference. The humanized antibody may be a human antibody in which somehypervariable region residues, and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483;5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023;6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

The humanized antibody may retain high affinity for UCH-L1 and otherfavorable biological properties. The humanized antibody may be preparedby a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available. Computer programs are available thatillustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristics, such as increased affinity forUCH-L1, is achieved. In general, the hypervariable region residues maybe directly and most substantially involved in influencing antigenbinding.

As an alternative to humanization, human antibodies (also referred toherein as “fully human antibodies”) can be generated. For example, it ispossible to isolate human antibodies from libraries via PROfusion and/oryeast related technologies. It is also possible to produce transgenicanimals (e.g., mice that are capable, upon immunization, of producing afull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. The humanized or fully humanantibodies may be prepared according to the methods described in U.S.Pat. Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517;6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;6,682,928; and 6,984,720, the contents each of which are hereinincorporated by reference.

e. Anti-UCH-L1 Antibodies

Anti-UCH-L1 antibodies may be generated using the techniques describedabove as well as using routine techniques known in the art. In someembodiments, the anti-UCH-L1 antibody may be an unconjugated UCH-L1antibody, such as UCH-L1 antibodies available from United StateBiological (Catalog Number: 031320), Cell Signaling Technology (CatalogNumber: 3524), Sigma-Aldrich (Catalog Number: HPA005993), Santa CruzBiotechnology, Inc. (Catalog Numbers: sc-58593 or sc-58594), R&D Systems(Catalog Number: MAB6007), Novus Biologicals (Catalog Number:NB600-1160), Biorbyt (Catalog Number: orb33715), Enzo Life Sciences,Inc. (Catalog Number: ADI-905-520-1), Bio-Rad (Catalog Number:VMA00004), BioVision (Catalog Number: 6130-50), Abcam (Catalog Numbers:ab75275 or ab104938), Invitrogen Antibodies (Catalog Numbers: 480012),ThermoFisher Scientific (Catalog Numbers: MA1-46079, MA5-17235,MA1-90008, or MA1-83428), EMD Millipore (Catalog Number: MABN48), orSino Biological Inc. (Catalog Number: 50690-R011). The anti-UCH-L1antibody may be conjugated to a fluorophore, such as conjugated UCH-L1antibodies available from BioVision (Catalog Number: 6960-25) or AvivaSystems Biology (Cat. Nos. OAAF01904-FITC).

7. Methods for Measuring the Level of GFAP

In the methods described above, GFAP levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, or protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of GFAP includes contacting thesample with a first specific binding member and second specific bindingmember. In some embodiments the first specific binding member is acapture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of GFAP includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., GFAP-capture antibody), which bindsto an epitope on GFAP or GFAP fragment to form a capture antibody-GFAPantigen complex (e.g., GFAP-capture antibody-GFAP antigen complex), and(2) a detection antibody (e.g., GFAP-detection antibody), which includesa detectable label and binds to an epitope on GFAP that is not bound bythe capture antibody, to form a GFAP antigen-detection antibody complex(e.g., GFAP antigen-GFAP-detection antibody complex), such that acapture antibody-GFAP antigen-detection antibody complex (e.g.,GFAP-capture antibody-GFAP antigen-GFAP-detection antibody complex) isformed, and measuring the amount or concentration of GFAP in the samplebased on the signal generated by the detectable label in the captureantibody-GFAP antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a GFAP antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example, the Abbott Laboratoriesimmunoassay analyzer ARCHITECT®, and other core laboratory instruments)other than a point-of-care device, may be capable of measuring levels ofGFAP in a sample higher or greater than 25,000 pg/mL.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety

8. GFAP Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to Glial fibrillary acidic protein (“GFAP”) (orfragments thereof), referred to as “GFAP antibody.” The GFAP antibodiescan be used to assess the GFAP status as a measure of traumatic braininjury, detect the presence of GFAP in a sample, quantify the amount ofGFAP present in a sample, or detect the presence of and quantify theamount of GFAP in a sample.

a. Glial Fibrillary Acidic Protein (GFAP)

Glial fibrillary acidic protein (GFAP) is a 50 kDa intracytoplasmicfilamentous protein that constitutes a portion of the cytoskeleton inastrocytes, and it has proved to be the most specific marker for cellsof astrocytic origin. GFAP protein is encoded by the GFAP gene inhumans. GFAP is the principal intermediate filament of matureastrocytes. In the central rod domain of the molecule, GFAP sharesconsiderable structural homology with the other intermediate filaments.GFAP is involved in astrocyte motility and shape by providing structuralstability to astrocytic processes. Glial fibrillary acidic protein andits breakdown products (GFAP-BDP) are brain-specific proteins releasedinto the blood as part of the pathophysiological response aftertraumatic brain injury (TBI). Following injury to the human CNS causedby trauma, genetic disorders, or chemicals, astrocytes proliferate andshow extensive hypertrophy of the cell body and processes, and GFAP ismarkedly upregulated. In contrast, with increasing astrocyte malignancy,there is a progressive loss of GFAP production. GFAP can also bedetected in Schwann cells, enteric glia cells, salivary gland neoplasms,metastasizing renal carcinomas, epiglottic cartilage, pituicytes,immature oligodendrocytes, papillary meningiomas, and myoepithelialcells of the breast.

Human GFAP may have the following amino acid sequence:

(SEQ ID NO: 2) MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.

The human GFAP may be a fragment or variant of SEQ ID NO: 2. Thefragment of GFAP may be between 5 and 400 amino acids, between 10 and400 amino acids, between 50 and 400 amino acids, between 60 and 400amino acids, between 65 and 400 amino acids, between 100 and 400 aminoacids, between 150 and 400 amino acids, between 100 and 300 amino acids,or between 200 and 300 amino acids in length. The fragment may comprisea contiguous number of amino acids from SEQ ID NO: 2. The human GFAPfragment or variant of SEQ ID NO: 2 may be a GFAP breakdown product(BDP). The GFAP BDP may be 38 kDa, 42 kDa (fainter 41 kDa), 47 kDa(fainter 45 kDa); 25 kDa (fainter 23 kDa); 19 kDa, or 20 kDa.

b. GFAP-Recognizing Antibody

The antibody is an antibody that binds to GFAP, a fragment thereof, anepitope of GFAP, or a variant thereof. The antibody may be a fragment ofthe anti-GFAP antibody or a variant or a derivative thereof. Theantibody may be a polyclonal or monoclonal antibody. The antibody may bea chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, a fully human antibodyor an antibody fragment, such as a Fab fragment, or a mixture thereof.Antibody fragments or derivatives may comprise F(ab′)₂, Fv or scFvfragments. The antibody derivatives can be produced by peptidomimetics.Further, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies.

The anti-GFAP antibodies may be a chimeric anti-GFAP or humanizedanti-GFAP antibody. In one embodiment, both the humanized antibody andchimeric antibody are monovalent. In one embodiment, both the humanizedantibody and chimeric antibody comprise a single Fab region linked to anFc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-GFAP antibodies. For example, libraries ofnaïve human single chain variable fragments (scFv) may be used to selecthuman anti-GFAP antibodies. Transgenic animals may be used to expresshuman antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

(1) Epitope

The antibody may immunospecifically bind to GFAP (SEQ ID NO: 2), afragment thereof, or a variant thereof. The antibody mayimmunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

c. Antibody Preparation/Production

Antibodies may be prepared by any of a variety of techniques, includingthose well known to those skilled in the art. In general, antibodies canbe produced by cell culture techniques, including the generation ofmonoclonal antibodies via conventional techniques, or via transfectionof antibody genes, heavy chains, and/or light chains into suitablebacterial or mammalian cell hosts, in order to allow for the productionof antibodies, wherein the antibodies may be recombinant. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies in either prokaryoticor eukaryotic host cells, expression of antibodies in eukaryotic cellsis preferable, and most preferable in mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesinclude Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody. Recombinant DNA technology may also be used to remove some, orall, of the DNA encoding either or both of the light and heavy chainsthat is not necessary for binding to the antigens of interest. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies. In addition, bifunctional antibodies maybe produced in which one heavy and one light chain are an antibody(i.e., binds human GFAP) and the other heavy and light chain arespecific for an antigen other than human GFAP by crosslinking anantibody to a second antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the method of synthesizing a recombinant antibodymay be by culturing a host cell in a suitable culture medium until arecombinant antibody is synthesized. The method can further compriseisolating the recombinant antibody from the culture medium.

Methods of preparing monoclonal antibodies involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Such cell lines may be produced from spleen cells obtainedfrom an immunized animal. The animal may be immunized with GFAP or afragment and/or variant thereof. The peptide used to immunize the animalmay comprise amino acids encoding human Fc, for example the fragmentcrystallizable region or tail region of human antibody. The spleen cellsmay then be immortalized by, for example, fusion with a myeloma cellfusion partner. A variety of fusion techniques may be employed. Forexample, the spleen cells and myeloma cells may be combined with anonionic detergent for a few minutes and then plated at low density on aselective medium that supports that growth of hybrid cells, but notmyeloma cells. One such technique uses hypoxanthine, aminopterin,thymidine (HAT) selection. Another technique includes electrofusion.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity may be used.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. Affinity chromatography is an example ofa method that can be used in a process to purify the antibodies.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment, which comprises bothantigen-binding sites.

The Fv fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecules. TheFv fragment may be derived using recombinant techniques. The Fv fragmentincludes a non-covalent VH::VL heterodimer including an antigen-bindingsite that retains much of the antigen recognition and bindingcapabilities of the native antibody molecule.

The antibody, antibody fragment, or derivative may comprise a heavychain and a light chain complementarity determining region (“CDR”) set,respectively interposed between a heavy chain and a light chainframework (“FR”) set which provide support to the CDRs and define thespatial relationship of the CDRs relative to each other. The CDR set maycontain three hypervariable regions of a heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g.,as available from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) Biolnvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al.(1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.).; Gray etal. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134 (1994)).

An affinity matured antibody may be produced by any one of a number ofprocedures that are known in the art. For example, see Marks et al.,BioTechnology, 10: 779-783 (1992) describes affinity maturation by VHand VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

Antibody variants can also be prepared using delivering a polynucleotideencoding an antibody to a suitable host such as to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. These methods are known inthe art and are described for example in U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody variants also can be prepared by delivering a polynucleotide toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol.Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies can also beproduced using transgenic plants, according to known methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally, part or all of the non-humanor human CDR sequences are maintained while the non-human sequences ofthe variable and constant regions are replaced with human or other aminoacids.

Small antibody fragments may be diabodies having two antigen-bindingsites, wherein fragments comprise a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (VH VL). See for example, EP 404,097; WO 93/11161; and Hollingeret al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. See also,U.S. Pat. No. 6,632,926 to Chen et al. which is hereby incorporated byreference in its entirety and discloses antibody variants that have oneor more amino acids inserted into a hypervariable region of the parentantibody and a binding affinity for a target antigen which is at leastabout two fold stronger than the binding affinity of the parent antibodyfor the antigen.

The antibody may be a linear antibody. The procedure for making a linearantibody is known in the art and described in Zapata et al. (1995)Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pairof tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The antibodies may be recovered and purified from recombinant cellcultures by known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

It may be useful to detectably label the antibody. Methods forconjugating antibodies to these agents are known in the art. For thepurpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.They can be linked to a cytokine, to a ligand, to another antibody.Suitable agents for coupling to antibodies to achieve an anti-tumoreffect include cytokines, such as interleukin 2 (IL-2) and TumorNecrosis Factor (TNF); photosensitizers, for use in photodynamictherapy, including aluminum (III) phthalocyanine tetrasulfonate,hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131(131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re);antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinesecobra (naja naja atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestrictocin (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; ly207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

Antibody production via the use of hybridoma technology, the selectedlymphocyte antibody method (SLAM), transgenic animals, and recombinantantibody libraries is described in more detail below.

(1) Anti-GFAP Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is alsonoted that the term “monoclonal antibody” as used herein is not limitedto antibodies produced through hybridoma technology. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

Methods of generating monoclonal antibodies as well as antibodiesproduced by the method may comprise culturing a hybridoma cell secretingan antibody of the invention wherein, preferably, the hybridoma isgenerated by fusing splenocytes isolated from an animal, e.g., a rat ora mouse, immunized with GFAP with myeloma cells and then screening thehybridomas resulting from the fusion for hybridoma clones that secretean antibody able to bind a polypeptide of the invention. Briefly, ratscan be immunized with a GFAP antigen. In a preferred embodiment, theGFAP antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks; however, a single administration of thepolypeptide may also be used.

After immunization of an animal with a GFAP antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-GFAPantibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-GFAP antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen GFAP are detected in the rat serum, the rat spleen is harvestedand splenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example, cells from cellline SP20 available from the American Type Culture Collection (ATCC,Manassas, Va., US). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding GFAP. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing rats with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using GFAP, or a portion thereof,or a cell expressing GFAP. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunosorbent assay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in PCT Publication No. WO 00/37504.

Anti-GFAP antibody-producing hybridomas are selected, cloned, andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production, and desirable antibodycharacteristics. Hybridomas may be cultured and expanded in vivo insyngeneic animals, in animals that lack an immune system, e.g., nudemice, or in cell culture in vitro. Methods of selecting, cloning andexpanding hybridomas are well known to those of ordinary skill in theart.

In a preferred embodiment, hybridomas are rat hybridomas. In anotherembodiment, hybridomas are produced in a non-human, non-rat species suchas mice, sheep, pigs, goats, cattle, or horses. In yet another preferredembodiment, the hybridomas are human hybridomas, in which a humannon-secretory myeloma is fused with a human cell expressing an anti-GFAPantibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce two identical Fabfragments) or pepsin (to produce an F(ab′)₂ fragment). A F(ab′)₂fragment of an IgG molecule retains the two antigen-binding sites of thelarger (“parent”) IgG molecule, including both light chains (containingthe variable light chain and constant light chain regions), the CH1domains of the heavy chains, and a disulfide-forming hinge region of theparent IgG molecule. Accordingly, an F(ab′)₂ fragment is still capableof crosslinking antigen molecules like the parent IgG molecule.

(2) Anti-GFAP Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook etal., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals are screened using anantigen-specific hemolytic plaque assay, wherein the antigen GFAP, asubunit of GFAP, or a fragment thereof, is coupled to sheep red bloodcells using a linker, such as biotin, and used to identify single cellsthat secrete antibodies with specificity for GFAP. Followingidentification of antibody-secreting cells of interest, heavy- andlight-chain variable region cDNAs are rescued from the cells by reversetranscriptase-PCR (RT-PCR) and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example, by panning thetransfected cells to isolate cells expressing antibodies to GFAP. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation method. See, for example, PCTPublication No. WO 97/29131 and PCT Publication No. WO 00/56772.

(3) Anti-GFAP Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the invention, antibodies are produced byimmunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with a GFAP antigen. In an embodiment, thenon-human animal is a XENOMOUSE® transgenic mouse, an engineered mousestrain that comprises large fragments of the human immunoglobulin lociand is deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771;5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and6,130,364. See also PCT Publication Nos. WO 91/10741; WO 94/02602; WO96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human monoclonal antibodies.The XENOMOUSE® transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics, 15: 146-156 (1997),Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosuresof which are hereby incorporated by reference.

(4) Anti-GFAP Monoclonal Antibodies Using Recombinant Antibody Libraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired GFAP-binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, U.S. Pat. No. 5,223,409(Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCTPublication No. WO 91/17271 (Dower et al.); PCT Publication No. WO92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland etal.); PCT Publication No. WO 93/01288 (Breitling et al.); PCTPublication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO92/09690 (Garrard et al.); Fuchs et al., Bio/Technology, 9: 1369-1372(1991); Hay et al., Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse etal., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkinset al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature,352: 624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio/Technology, 9: 1373-1377 (1991);Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas etal., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); U.S. PatentApplication Publication No. 2003/0186374; and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

The recombinant antibody library may be from a subject immunized withGFAP, or a portion of GFAP. Alternatively, the recombinant antibodylibrary may be from a naive subject, i.e., one who has not beenimmunized with GFAP, such as a human antibody library from a humansubject who has not been immunized with human GFAP. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human GFAP to thereby select thoseantibodies that recognize GFAP. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for GFAP, such as thosethat dissociate from human GFAP with a particular K_(off) rate constant,the art-known method of surface plasmon resonance can be used to selectantibodies having the desired K_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hGFAP, such as those with a particular IC₅₀, standard methods knownin the art for assessing the inhibition of GFAP activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human GFAP. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. Such phage can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Phageexpressing an antigen binding domain that binds the antigen of interestcan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv, or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies include those disclosed in Brinkmannet al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al.,Advances in Immunology, 57: 191-280 (1994); PCT Publication No. WO92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743; and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al.,Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al.,Science, 240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the invention. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98/31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology is PROfusion display technology.

In another approach, the antibodies can also be generated using yeastdisplay methods known in the art. In yeast display methods, geneticmethods are used to tether antibody domains to the yeast cell wall anddisplay them on the surface of yeast. In particular, such yeast can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Examples ofyeast display methods that can be used to make the antibodies includethose disclosed in U.S. Pat. No. 6,699,658 (Wittrup et al.) incorporatedherein by reference.

d. Production of Recombinant GFAP Antibodies

Antibodies may be produced by any of a number of techniques known in theart. For example, expression from host cells, wherein expressionvector(s) encoding the heavy and light chains is (are) transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection, and the like. Although it ispossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J Mol. Biol., 159: 601-621 (1982), NSOmyeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody of this invention. Recombinant DNA technology may also be usedto remove some, or all, of the DNA encoding either or both of the lightand heavy chains that is not necessary for binding to the antigens ofinterest. The molecules expressed from such truncated DNA molecules arealso encompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention (i.e., binds human GFAP) and theother heavy and light chain are specific for an antigen other than humanGFAP by crosslinking an antibody of the invention to a second antibodyby standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

(1) Humanized Antibody

The humanized antibody may be an antibody or a variant, derivative,analog or portion thereof which immunospecifically binds to an antigenof interest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. The humanized antibody may befrom a non-human species antibody that binds the desired antigen havingone or more complementarity determining regions (CDRs) from thenon-human species and framework regions from a human immunoglobulinmolecule.

As used herein, the term “substantially” in the context of a CDR refersto a CDR having an amino acid sequence at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′,F(ab′)₂, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. According to one aspect, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or of a heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3, and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In one embodiment, suchmutations, however, will not be extensive. Usually, at least 90%, atleast 95%, at least 98%, or at least 99% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences.As used herein, the term “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence. As used herein, theterm “consensus immunoglobulin sequence” refers to the sequence formedfrom the most frequently occurring amino acids (or nucleotides) in afamily of related immunoglobulin sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In afamily of immunoglobulins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

The humanized antibody may be designed to minimize unwantedimmunological response toward rodent anti-human antibodies, which limitsthe duration and effectiveness of therapeutic applications of thosemoieties in human recipients. The humanized antibody may have one ormore amino acid residues introduced into it from a source that isnon-human. These non-human residues are often referred to as “import”residues, which are typically taken from a variable domain. Humanizationmay be performed by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. For example, see U.S.Pat. No. 4,816,567, the contents of which are herein incorporated byreference. The humanized antibody may be a human antibody in which somehypervariable region residues, and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483;5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023;6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

The humanized antibody may retain high affinity for GFAP and otherfavorable biological properties. The humanized antibody may be preparedby a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available. Computer programs are available thatillustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristics, such as increased affinity forGFAP, is achieved. In general, the hypervariable region residues may bedirectly and most substantially involved in influencing antigen binding.

As an alternative to humanization, human antibodies (also referred toherein as “fully human antibodies”) can be generated. For example, it ispossible to isolate human antibodies from libraries via PROfusion and/oryeast related technologies. It is also possible to produce transgenicanimals (e.g. mice that are capable, upon immunization, of producing afull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. The humanized or fully humanantibodies may be prepared according to the methods described in U.S.Pat. Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517;6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;6,682,928; and 6,984,720, the contents each of which are hereinincorporated by reference.

e. Anti-GFAP Antibodies

Anti-GFAP antibodies may be generated using the techniques describedabove as well as using routine techniques known in the art. In someembodiments, the anti-GFAP antibody may be an unconjugated GFAPantibody, such as GFAP antibodies available from Dako (Catalog Number:M0761), ThermoFisher Scientific (Catalog Numbers: MA5-12023, A-21282,13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377,MA1-06701, or MA1-20035), AbCam (Catalog Numbers: ab10062, ab4648,ab68428, ab33922, ab207165, ab190288, ab115898, or ab21837), EMDMillipore (Catalog Numbers: FCMAB257P, MAB360, MAB3402, 04-1031,04-1062, MAB5628), Santa Cruz (Catalog Numbers: sc-166481, sc-166458,sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343, orsc-33673), Sigma-Aldrich (Catalog Numbers: G3893 or G6171) or SinoBiological Inc. (Catalog Number: 100140-R012-50). The anti-GFAP antibodymay be conjugated to a fluorophore, such as conjugated GFAP antibodiesavailable from ThermoFisher Scientific (Catalog Numbers: A-21295 orA-21294), EMD Millipore (Catalog Numbers: MAB3402X, MAB3402B, MAB3402B,or MAB3402C3) or AbCam (Catalog Numbers; ab49874 or ab194325).

9. Variations on Methods

The disclosed methods of determining the presence or amount of analyteof interest (UCH-L1 and/or GFAP) present in a sample may be as describedherein. The methods may also be adapted in view of other methods foranalyzing analytes. Examples of well-known variations include, but arenot limited to, immunoassay, such as sandwich immunoassay (e.g.,monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonalsandwich immunoassays, including enzyme detection (enzyme immunoassay(EIA) or enzyme-linked immunosorbent assay (ELISA), competitiveinhibition immunoassay (e.g., forward and reverse), enzyme multipliedimmunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc.

a. Immunoassay

The analyte of interest, and/or peptides of fragments thereof (e.g.,UCH-L1 and/or GFAP, and/or peptides or fragments thereof, i.e., UCH-L1and/or GFAP fragments), may be analyzed using UCH-L1 and/or GFAPantibodies in an immunoassay. The presence or amount of analyte (e.g.,UCH-L1 and/or GFAP) can be determined using antibodies and detectingspecific binding to the analyte (e.g., UCH-L1 and/or GFAP). For example,the antibody, or antibody fragment thereof, may specifically bind to theanalyte (e.g., UCH-L1 and/or GFAP). If desired, one or more of theantibodies can be used in combination with one or more commerciallyavailable monoclonal/polyclonal antibodies. Such antibodies areavailable from companies such as R&D Systems, Inc. (Minneapolis, Minn.)and Enzo Life Sciences International, Inc. (Plymouth Meeting, Pa.).

The presence or amount of analyte (e.g., UCH-L1 and/or GFAP) present ina body sample may be readily determined using an immunoassay, such assandwich immunoassay (e.g., monoclonal-monoclonal sandwich immunoassays,monoclonal-polyclonal sandwich immunoassays, including radioisotopedetection (radioimmunoassay (RIA)) and enzyme detection (enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g.,Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)). An exampleof a point-of-care device that can be used is i-STAT® (Abbott,Laboratories, Abbott Park, Ill.). Other methods that can be used includea chemiluminescent microparticle immunoassay, in particular oneemploying the ARCHITECT® automated analyzer (Abbott Laboratories, AbbottPark, Ill.), as an example. Other methods include, for example, massspectrometry, and immunohistochemistry (e.g., with sections from tissuebiopsies), using anti-analyte (e.g., anti-UCH-L1 and/or anti-GFAP)antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) orantibody fragments thereof against analyte (e.g., UCH-L1 and/or GFAP).Other methods of detection include those described in, for example, U.S.Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124;5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526;5,525,524; and 5,480,792, each of which is hereby incorporated byreference in its entirety. Specific immunological binding of theantibody to the analyte (e.g., UCH-L1 and/or GFAP) can be detected viadirect labels, such as fluorescent or luminescent tags, metals andradionuclides attached to the antibody or via indirect labels, such asalkaline phosphatase or horseradish peroxidase.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, the surface of an assay plate (such as microtiter wells),pieces of a solid substrate material, and the like. An assay strip canbe prepared by coating the antibody or plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

A homogeneous format may be used. For example, after the test sample isobtained from a subject, a mixture is prepared. The mixture contains thetest sample being assessed for analyte (e.g., UCH-L1 and/or GFAP), afirst specific binding partner, and a second specific binding partner.The order in which the test sample, the first specific binding partner,and the second specific binding partner are added to form the mixture isnot critical. The test sample is simultaneously contacted with the firstspecific binding partner and the second specific binding partner. Insome embodiments, the first specific binding partner and any UCH-L1and/or GFAP contained in the test sample may form a first specificbinding partner-analyte (e.g., UCH-L1 and/or GFAP)-antigen complex andthe second specific binding partner may form a first specific bindingpartner-analyte of interest (e.g., UCH-L1 and/or GFAP)-second specificbinding partner complex. In some embodiments, the second specificbinding partner and any UCH-L1 and/or GFAP contained in the test samplemay form a second specific binding partner-analyte (e.g.,UCH-L1)-antigen complex and the first specific binding partner may forma first specific binding partner-analyte of interest (e.g., UCH-L1and/or GFAP)-second specific binding partner complex. The first specificbinding partner may be an anti-analyte antibody (e.g., anti-UCH-L1antibody that binds to an epitope having an amino acid sequencecomprising at least three contiguous (3) amino acids of SEQ ID NO: 1 oranti-GFAP antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 2). The second specific binding partner may be an anti-analyteantibody (e.g., anti-UCH-L1 antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous (3) amino acidsof SEQ ID NO: 1 or anti-GFAP antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous (3) amino acidsof SEQ ID NO: 2). Moreover, the second specific binding partner islabeled with or contains a detectable label as described above.

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for analyte (e.g., UCH-L1 and/orGFAP) and a first specific binding partner, wherein the first specificbinding partner and any UCH-L1 and/or GFAP contained in the test sampleform a first specific binding partner-analyte (e.g., UCH-L1 and/orGFAP)-antigen complex. The first specific binding partner may be ananti-analyte antibody (e.g., anti-UCH-L1 antibody that binds to anepitope having an amino acid sequence comprising at least threecontiguous (3) amino acids of SEQ ID NO: 1 or anti-GFAP antibody thatbinds to an epitope having an amino acid sequence comprising at leastthree contiguous (3) amino acids of SEQ ID NO: 2). The order in whichthe test sample and the first specific binding partner are added to formthe mixture is not critical.

The first specific binding partner may be immobilized on a solid phase.The solid phase used in the immunoassay (for the first specific bindingpartner and, optionally, the second specific binding partner) can be anysolid phase known in the art, such as, but not limited to, a magneticparticle, a bead, a test tube, a microtiter plate, a cuvette, amembrane, a scaffolding molecule, a film, a filter paper, a disc, and achip. In those embodiments where the solid phase is a bead, the bead maybe a magnetic bead or a magnetic particle. Magnetic beads/particles maybe ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄(or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The solid support on which the firstspecific binding member is immobilized may be stored in dry form or in aliquid. The magnetic beads may be subjected to a magnetic field prior toor after contacting with the sample with a magnetic bead on which thefirst specific binding member is immobilized.

After the mixture containing the first specific binding partner-analyte(e.g., UCH-L1 or GFAP) antigen complex is formed, any unbound analyte(e.g., UCH-L1 and/or GFAP) is removed from the complex using anytechnique known in the art. For example, the unbound analyte (e.g.,UCH-L1 and/or GFAP) can be removed by washing. Desirably, however, thefirst specific binding partner is present in excess of any analyte(e.g., UCH-L1 and/or GFAP) present in the test sample, such that allanalyte (e.g., UCH-L1 and/or GFAP) that is present in the test sample isbound by the first specific binding partner.

After any unbound analyte (e.g., UCH-L1 and/or GFAP) is removed, asecond specific binding partner is added to the mixture to form a firstspecific binding partner-analyte of interest (e.g., UCH-L1 and/orGFAP)-second specific binding partner complex. The second specificbinding partner may be an anti-analyte antibody (e.g., anti-UCH-L1antibody that binds to an epitope having an amino acid sequencecomprising at least three contiguous (3) amino acids of SEQ ID NO: 1 oranti-GFAP antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 2). Moreover, the second specific binding partner is labeled with orcontains a detectable label as described above.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles (such as a magnetic bead), latex particles or modified surfacelatex particles, polymer or polymer film, plastic or plastic film,planar substrate, the surface of an assay plate (such as microtiterwells), pieces of a solid substrate material, and the like. An assaystrip can be prepared by coating the antibody or plurality of antibodiesin an array on a solid support. This strip can then be dipped into thetest sample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

(1) Sandwich Immunoassay

A sandwich immunoassay measures the amount of antigen between two layersof antibodies (i.e., at least one capture antibody) and a detectionantibody (i.e., at least one detection antibody). The capture antibodyand the detection antibody bind to different epitopes on the antigen,e.g., analyte of interest such as UCH-L1 and/or GFAP. Desirably, bindingof the capture antibody to an epitope does not interfere with binding ofthe detection antibody to an epitope. Either monoclonal or polyclonalantibodies may be used as the capture and detection antibodies in thesandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte (e.g., UCH-L1 and/or GFAP) in a test sample. More specifically,the at least two antibodies bind to certain epitopes of analyte (e.g.,UCH-L1 and/or GFAP) forming an immune complex which is referred to as a“sandwich”. One or more antibodies can be used to capture the analyte(e.g., UCH-L1 and/or GFAP) in the test sample (these antibodies arefrequently referred to as a “capture” antibody or “capture” antibodies)and one or more antibodies is used to bind a detectable (namely,quantifiable) label to the sandwich (these antibodies are frequentlyreferred to as the “detection” antibody or “detection” antibodies). In asandwich assay, the binding of an antibody to its epitope desirably isnot diminished by the binding of any other antibody in the assay to itsrespective epitope. Antibodies are selected so that the one or morefirst antibodies brought into contact with a test sample suspected ofcontaining analyte (e.g., UCH-L1 and/or GFAP) do not bind to all or partof an epitope recognized by the second or subsequent antibodies, therebyinterfering with the ability of the one or more second detectionantibodies to bind to the analyte (e.g., UCH-L1 and/or GFAP).

The antibodies may be used as a first antibody in said immunoassay. Theantibody immunospecifically binds to epitopes on analyte (e.g., UCH-L1and/or GFAP). In addition to the antibodies of the present invention,said immunoassay may comprise a second antibody that immunospecificallybinds to epitopes that are not recognized or bound by the firstantibody.

A test sample suspected of containing analyte (e.g., UCH-L1 and/or GFAP)can be contacted with at least one first capture antibody (orantibodies) and at least one second detection antibodies eithersimultaneously or sequentially. In the sandwich assay format, a testsample suspected of containing analyte (e.g., UCH-L1 and/or GFAP) isfirst brought into contact with the at least one first capture antibodythat specifically binds to a particular epitope under conditions whichallow the formation of a first antibody-analyte (e.g., UCH-L1 and/orGFAP) antigen complex. If more than one capture antibody is used, afirst multiple capture antibody-UCH-L1 and/or GFAP antigen complex isformed. In a sandwich assay, the antibodies, preferably, the at leastone capture antibody, are used in molar excess amounts of the maximumamount of analyte (e.g., UCH-L1 and/or GFAP) expected in the testsample. For example, from about 5 pg/mL to about 1 mg/mL of antibody perml of microparticle coating buffer may be used.

i. Anti-UCH-L1 Capture Antibody

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one first capture antibody can bebound to a solid support which facilitates the separation the firstantibody-analyte (e.g., UCH-L1 and/or GFAP) complex from the testsample. Any solid support known in the art can be used, including butnot limited to, solid supports made out of polymeric materials in theforms of wells, tubes, or beads (such as a microparticle). The antibody(or antibodies) can be bound to the solid support by adsorption, bycovalent bonding using a chemical coupling agent or by other means knownin the art, provided that such binding does not interfere with theability of the antibody to bind analyte (e.g., UCH-L1 and/or GFAP).Moreover, if necessary, the solid support can be derivatized to allowreactivity with various functional groups on the antibody. Suchderivatization requires the use of certain coupling agents such as, butnot limited to, maleic anhydride, N-hydroxysuccinimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte (e.g., UCH-L1and/or GFAP) is incubated in order to allow for the formation of a firstcapture antibody (or multiple antibody)-analyte (e.g., UCH-L1 and/orGFAP) complex. The incubation can be carried out at a pH of from about4.5 to about 10.0, at a temperature of from about 2° C. to about 45° C.,and for a period from at least about one (1) minute to about eighteen(18) hours, from about 2-6 minutes, from about 7-12 minutes, from about5-15 minutes, or from about 3-4 minutes.

ii. Detection Antibody

After formation of the first/multiple capture antibody-analyte (e.g.,UCH-L1 and/or GFAP) complex, the complex is then contacted with at leastone second detection antibody (under conditions that allow for theformation of a first/multiple antibody-analyte (e.g., UCH-L1 and/orGFAP) antigen-second antibody complex). In some embodiments, the testsample is contacted with the detection antibody simultaneously with thecapture antibody. If the first antibody-analyte (e.g., UCH-L1 and/orGFAP) complex is contacted with more than one detection antibody, then afirst/multiple capture antibody-analyte (e.g., UCH-L1 and/orGFAP)-multiple antibody detection complex is formed. As with firstantibody, when the at least second (and subsequent) antibody is broughtinto contact with the first antibody-analyte (e.g., UCH-L1 and/or GFAP)complex, a period of incubation under conditions similar to thosedescribed above is required for the formation of the first/multipleantibody-analyte (e.g., UCH-L1 and/or GFAP)-second/multiple antibodycomplex. Preferably, at least one second antibody contains a detectablelabel. The detectable label can be bound to the at least one secondantibody prior to, simultaneously with or after the formation of thefirst/multiple antibody-analyte (e.g., UCH-L1 and/orGFAP)-second/multiple antibody complex. Any detectable label known inthe art can be used.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith an acridinium compound, detectably labeled first specific bindingpartner-antigen (e.g., UCH-L1 and/or GFAP) complexes form.Alternatively, if a second specific binding partner is used and thesecond specific binding partner is detectably labeled with an acridiniumcompound, detectably labeled first specific binding partner-analyte(e.g., UCH-L1 and/or GFAP)-second specific binding partner complexesform. Any unbound specific binding partner, whether labeled orunlabeled, can be removed from the mixture using any technique known inthe art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture before, simultaneously with, or after theaddition of an above-described acridinium compound. Hydrogen peroxidecan be generated in situ in a number of ways such as would be apparentto one skilled in the art.

Alternatively, a source of hydrogen peroxide can be simply added to themixture. For example, the source of the hydrogen peroxide can be one ormore buffers or other solutions that are known to contain hydrogenperoxide. In this regard, a solution of hydrogen peroxide can simply beadded.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte (e.g., UCH-L1 and/or GFAP)is generated. The basic solution contains at least one base and has a pHgreater than or equal to 10, preferably, greater than or equal to 12.Examples of basic solutions include, but are not limited to, sodiumhydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide,magnesium hydroxide, sodium carbonate, sodium bicarbonate, calciumhydroxide, calcium carbonate, and calcium bicarbonate. The amount ofbasic solution added to the sample depends on the concentration of thebasic solution. Based on the concentration of the basic solution used,one skilled in the art can easily determine the amount of basic solutionto add to the sample. Other labels other than chemiluminescent labelscan be employed. For instance, enzymatic labels (including but notlimited to alkaline phosphatase) can be employed.

The chemiluminescent signal, or other signal, that is generated can bedetected using routine techniques known to those skilled in the art.Based on the intensity of the signal generated, the amount of analyte ofinterest (e.g., UCH-L1 and/or GFAP) in the sample can be quantified.Specifically, the amount of analyte (e.g., UCH-L1 and/or GFAP) in thesample is proportional to the intensity of the signal generated. Theamount of analyte (e.g., UCH-L1 and/or GFAP) present can be quantifiedby comparing the amount of light generated to a standard curve foranalyte (e.g., UCH-L1 and/or GFAP) or by comparison to a referencestandard. The standard curve can be generated using serial dilutions orsolutions of known concentrations of analyte (e.g., UCH-L1 and/or GFAP)by mass spectroscopy, gravimetric methods, and other techniques known inthe art.

(2) Forward Competitive Inhibition Assay

In a forward competitive format, an aliquot of labeled analyte ofinterest (e.g., analyte (e.g., UCH-L1 and/or GFAP) having a fluorescentlabel, a tag attached with a cleavable linker, etc.) of a knownconcentration is used to compete with analyte of interest (e.g., UCH-L1and/or GFAP) in a test sample for binding to analyte of interestantibody (e.g., UCH-L1 and/or GFAP antibody).

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and a labeled analyte of interest,analyte of interest fragment or analyte of interest variant thereof. Theanalyte of interest peptide, analyte of interest fragment or analyte ofinterest variant can be labeled with any detectable label, including adetectable label comprised of tag attached with a cleavable linker. Inthis assay, the antibody can be immobilized on to a solid support.Alternatively, the antibody can be coupled to an antibody, such as anantispecies antibody, that has been immobilized on a solid support, suchas a microparticle or planar substrate.

The labeled analyte of interest, the test sample and the antibody areincubated under conditions similar to those described above inconnection with the sandwich assay format. Two different species ofantibody-analyte of interest complexes may then be generated.Specifically, one of the antibody-analyte of interest complexesgenerated contains a detectable label (e.g., a fluorescent label, etc.)while the other antibody-analyte of interest complex does not contain adetectable label. The antibody-analyte of interest complex can be, butdoes not have to be, separated from the remainder of the test sampleprior to quantification of the detectable label. Regardless of whetherthe antibody-analyte of interest complex is separated from the remainderof the test sample, the amount of detectable label in theantibody-analyte of interest complex is then quantified. Theconcentration of analyte of interest (such as membrane-associatedanalyte of interest, soluble analyte of interest, fragments of solubleanalyte of interest, variants of analyte of interest(membrane-associated or soluble analyte of interest) or any combinationsthereof) in the test sample can then be determined, e.g., as describedabove.

(3) Reverse Competitive Inhibition Assay

In a reverse competition assay, an immobilized analyte of interest(e.g., UCH-L1 and/or GFAP) can either be sequentially or simultaneouslycontacted with a test sample and at least one labeled antibody.

The analyte of interest can be bound to a solid support, such as thesolid supports discussed above in connection with the sandwich assayformat.

The immobilized analyte of interest, test sample and at least onelabeled antibody are incubated under conditions similar to thosedescribed above in connection with the sandwich assay format. Twodifferent species analyte of interest-antibody complexes are thengenerated. Specifically, one of the analyte of interest-antibodycomplexes generated is immobilized and contains a detectable label(e.g., a fluorescent label, etc.) while the other analyte ofinterest-antibody complex is not immobilized and contains a detectablelabel. The non-immobilized analyte of interest-antibody complex and theremainder of the test sample are removed from the presence of theimmobilized analyte of interest-antibody complex through techniquesknown in the art, such as washing. Once the non-immobilized analyte ofinterest antibody complex is removed, the amount of detectable label inthe immobilized analyte of interest-antibody complex is then quantifiedfollowing cleavage of the tag. The concentration of analyte of interestin the test sample can then be determined by comparing the quantity ofdetectable label as described above.

(4) One-Step Immunoassay or “Capture on the Fly” Assay

In a capture on the fly immunoassay, a solid substrate is pre-coatedwith an immobilization agent. The capture agent, the analyte (e.g.,UCH-L1 and/or GFAP) and the detection agent are added to the solidsubstrate together, followed by a wash step prior to detection. Thecapture agent can bind the analyte (e.g., UCH-L1 and/or GFAP) andcomprises a ligand for an immobilization agent. The capture agent andthe detection agents may be antibodies or any other moiety capable ofcapture or detection as described herein or known in the art. The ligandmay comprise a peptide tag and an immobilization agent may comprise ananti-peptide tag antibody. Alternately, the ligand and theimmobilization agent may be any pair of agents capable of bindingtogether so as to be employed for a capture on the fly assay (e.g.,specific binding pair, and others such as are known in the art). Morethan one analyte may be measured. In some embodiments, the solidsubstrate may be coated with an antigen and the analyte to be analyzedis an antibody.

In certain other embodiments, in a one-step immunoassay or “capture onthe fly”, a solid support (such as a microparticle) pre-coated with animmobilization agent (such as biotin, streptavidin, etc.) and at least afirst specific binding member and a second specific binding member(which function as capture and detection reagents, respectively) areused. The first specific binding member comprises a ligand for theimmobilization agent (for example, if the immobilization agent on thesolid support is streptavidin, the ligand on the first specific bindingmember may be biotin) and also binds to the analyte of interest (e.g.,UCH-L1 and/or GFAP). The second specific binding member comprises adetectable label and binds to an analyte of interest (e.g., UCH-L1and/or GFAP). The solid support and the first and second specificbinding members may be added to a test sample (either sequentially orsimultaneously). The ligand on the first specific binding member bindsto the immobilization agent on the solid support to form a solidsupport/first specific binding member complex. Any analyte of interestpresent in the sample binds to the solid support/first specific bindingmember complex to form a solid support/first specific bindingmember/analyte complex. The second specific binding member binds to thesolid support/first specific binding member/analyte complex and thedetectable label is detected. An optional wash step may be employedbefore the detection. In certain embodiments, in a one-step assay morethan one analyte may be measured. In certain other embodiments, morethan two specific binding members can be employed. In certain otherembodiments, multiple detectable labels can be added. In certain otherembodiments, multiple analytes of interest can be detected, or theiramounts, levels or concentrations, measured, determined or assessed.

The use of a capture on the fly assay can be done in a variety offormats as described herein, and known in the art. For example theformat can be a sandwich assay such as described above, but alternatelycan be a competition assay, can employ a single specific binding member,or use other variations such as are known.

10. Other Factors

The methods of diagnosing, prognosticating, and/or assessing, asdescribed above, can further include using other factors for thediagnosis, prognostication, and assessment. In some embodiments,traumatic brain injury may be diagnosed using the Glasgow Coma Scale orthe Extended Glasgow Outcome Scale (GOSE). Other tests, scales orindices can also be used either alone or in combination with the GlasgowComa Scale. An example is the Ranchos Los Amigos Scale. The Ranchos LosAmigos Scale measures the levels of awareness, cognition, behavior andinteraction with the environment. The Ranchos Los Amigos Scale includes:Level I: No Response; Level II: Generalized Response; Level III:Localized Response; Level IV: Confused-agitated; Level V:Confused-inappropriate; Level VI: Confused-appropriate; Level VII:Automatic-appropriate; and Level VIII: Purposeful-appropriate.

11. Samples

In some embodiments, the sample is obtained after the human subjectsustained an injury to the head caused by physical shaking, blunt impactby an external mechanical or other force that results in a closed oropen head trauma, one or more falls, explosions or blasts or other typesof blunt force trauma. In some embodiments, the sample is obtained afterthe human subject has ingested or been exposed to a chemical, toxin orcombination of a chemical and toxin. Examples of such chemicals and/ortoxins include, fires, molds, asbestos, pesticides and insecticides,organic solvents, paints, glues, gases (such as carbon monoxide,hydrogen sulfide, and cyanide), organic metals (such as methyl mercury,tetraethyl lead and organic tin) and/or one or more drugs of abuse. Insome embodiments, the sample is obtained from a human subject thatsuffers from an autoimmune disease, a metabolic disorder, a brain tumor,hypoxia, one or more viruses, meningitis, hydrocephalus or combinationsthereof.

In yet another embodiment, the methods described herein use samples thatalso can be used to determine whether or not a subject has or is at riskof developing mild traumatic brain injury by determining the levels ofUCH-L1 and/or GFAP in a subject using the anti-UCH-L1 and/or anti-GFAPantibodies described below, or antibody fragments thereof. Thus, inparticular embodiments, the disclosure also provides a method fordetermining whether a subject having, or at risk for, traumatic braininjuries, discussed herein and known in the art, is a candidate fortherapy or treatment. Generally, the subject is at least one who: (i)has experienced an injury to the head; (ii) ingested and/or been exposedto one or more chemicals and/or toxins; (iii) suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, one or moreviruses, meningitis, hydrocephalus or suffers from any combinationsthereof; or (iv) any combinations of (i)-(iii); or, who has actuallybeen diagnosed as having, or being at risk for TBI (such as, forexample, subjects suffering from an autoimmune disease, a metabolicdisorder, a brain tumor, hypoxia, one or more viruses, meningitis,hydrocephalus or combinations thereof), and/or who demonstrates anunfavorable (i.e., clinically undesirable) concentration or amount ofUCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragment, as described herein.

a. Test or Biological Sample

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing UCH-L1 and/or GFAP.The sample may be derived from any suitable source. In some cases, thesample may comprise a liquid, fluent particulate solid, or fluidsuspension of solid particles. In some cases, the sample may beprocessed prior to the analysis described herein. For example, thesample may be separated or purified from its source prior to analysis;however, in certain embodiments, an unprocessed sample containing UCH-L1and/or GFAP may be assayed directly. In a particular example, the sourceof UCH-L1 and/or GFAP is a human bodily substance (e.g., bodily fluid,blood such as whole blood, serum, plasma, urine, saliva, sweat, sputum,semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitialfluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or thelike). Tissues may include, but are not limited to skeletal muscletissue, liver tissue, lung tissue, kidney tissue, myocardial tissue,brain tissue, bone marrow, cervix tissue, skin, etc. The sample may be aliquid sample or a liquid extract of a solid sample. In certain cases,the source of the sample may be an organ or tissue, such as a biopsysample, which may be solubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or thelike. In some cases, the volume of the fluid sample is between about0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, betweenabout 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of UCH-L1 and/or GFAP is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,prior to use. In other cases, the fluid sample is not diluted prior touse in an assay.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source of UCH-L1 and/or GFAP is a human bodyfluid (e.g., blood, serum), the fluid may be concentrated byprecipitation, evaporation, filtration, centrifugation, or a combinationthereof. A fluid sample may be concentrated about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold,about 100-fold, or greater, prior to use.

b. Controls

It may be desirable to include a control sample. The control sample maybe analyzed concurrently with the sample from the subject as describedabove. The results obtained from the subject sample can be compared tothe results obtained from the control sample. Standard curves may beprovided, with which assay results for the sample may be compared. Suchstandard curves present levels of marker as a function of assay units,i.e., fluorescent signal intensity, if a fluorescent label is used.Using samples taken from multiple donors, standard curves can beprovided for reference levels of the UCH-L1 and/or GFAP in normalhealthy tissue, as well as for “at-risk” levels of the UCH-L1 and/orGFAP in tissue taken from donors, who may have one or more of thecharacteristics set forth above.

Thus, in view of the above, a method for determining the presence,amount, or concentration of UCH-L1 and/or GFAP in a test sample isprovided. The method comprises assaying the test sample for UCH-L1and/or GFAP by an immunoassay, for example, employing at least onecapture antibody that binds to an epitope on UCH-L1 and/or GFAP and atleast one detection antibody that binds to an epitope on UCH-L1 and/orGFAP which is different from the epitope for the capture antibody andoptionally includes a detectable label, and comprising comparing asignal generated by the detectable label as a direct or indirectindication of the presence, amount or concentration of UCH-L1 and/orGFAP in the test sample to a signal generated as a direct or indirectindication of the presence, amount or concentration of UCH-L1 and/orGFAP in a calibrator. The calibrator is optionally, and is preferably,part of a series of calibrators in which each of the calibrators differsfrom the other calibrators in the series by the concentration of UCH-L1and/or GFAP.

12. Kit

Provided herein is a kit, which may be used for assaying or assessing atest sample for UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragment. Thekit comprises at least one component for assaying the test sample forUCH-L1 and/or GFAP instructions for assaying the test sample for UCH-L1and/or GFAP. For example, the kit can comprise instructions for assayingthe test sample for UCH-L1 and/or GFAP by immunoassay, e.g.,chemiluminescent microparticle immunoassay. Instructions included inkits can be affixed to packaging material or can be included as apackage insert. While the instructions are typically written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this disclosure. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

The at least one component may include at least one compositioncomprising one or more isolated antibodies or antibody fragments thereofthat specifically bind to UCH-L1 and/or GFAP. The antibody may be aUCH-L1 and/or GFAP capture antibody and/or a UCH-L1 and/or GFAPdetection antibody.

Alternatively or additionally, the kit can comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized, UCH-L1 and/or GFAP,and/or at least one container (e.g., tube, microtiter plates or strips,which can be already coated with an anti-UCH-L1 and/or GFAP monoclonalantibody) for conducting the assay, and/or a buffer, such as an assaybuffer or a wash buffer, either one of which can be provided as aconcentrated solution, a substrate solution for the detectable label(e.g., an enzymatic label), or a stop solution. Preferably, the kitcomprises all components, i.e., reagents, standards, buffers, diluents,etc., which are necessary to perform the assay. The instructions alsocan include instructions for generating a standard curve.

The kit may further comprise reference standards for quantifying UCH-L1and/or GFAP. The reference standards may be employed to establishstandard curves for interpolation and/or extrapolation of UCH-L1 and/orGFAP concentrations. The reference standards may include a high UCH-L1and/or GFAP concentration level, for example, about 100000 pg/mL, about125000 pg/mL, about 150000 pg/mL, about 175000 pg/mL, about 200000pg/mL, about 225000 pg/mL, about 250000 pg/mL, about 275000 pg/mL, orabout 300000 pg/mL; a medium UCH-L1 and/or GFAP concentration level, forexample, about 25000 pg/mL, about 40000 pg/mL, about 45000 pg/mL, about50000 pg/mL, about 55000 pg/mL, about 60000 pg/mL, about 75000 pg/mL orabout 100000 pg/mL; and/or a low UCH-L1 and/or GFAP concentration level,for example, about 1 pg/mL, about 5 pg/mL, about 10 pg/mL, about 12.5pg/mL, about 15 pg/mL, about 20 pg/mL, about 25 pg/mL, about 30 pg/mL,about 35 pg/mL, about 40 pg/mL, about 45 pg/mL, about 50 pg/mL, about 55pg/mL, about 60 pg/mL, about 65 pg/mL, about 70 pg/mL, about 75 pg/mL,about 80 pg/mL, about 85 pg/mL, about 90 pg/mL, about 95 pg/mL, or about100 pg/mL.

Any antibodies, which are provided in the kit, such as recombinantantibodies specific for UCH-L1 and/or GFAP, can incorporate a detectablelabel, such as a fluorophore, radioactive moiety, enzyme, biotin/avidinlabel, chromophore, chemiluminescent label, or the like, or the kit caninclude reagents for labeling the antibodies or reagents for detectingthe antibodies (e.g.; detection antibodies) and/or for labeling theanalytes (e.g., UCH-L1 and/or GFAP) or reagents for detecting theanalyte (e.g., UCH-L1 and/or GFAP). The antibodies, calibrators, and/orcontrols can be provided in separate containers or pre-dispensed into anappropriate assay format, for example, into microtiter plates,

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays,

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine, whole blood, plasma, or serum sample). Whereappropriate, the kit optionally also can contain reaction vessels,mixing vessels, and other components that facilitate the preparation ofreagents or the test sample. The kit can also include one or moreinstrument for assisting with obtaining a test sample, such as asyringe, pipette, forceps, measured spoon, or the like.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper,disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of traumatic brain injury or disorder.

a. Adaptation of Kit and Method

The kit (or components thereof), as well as the method for assessing ordetermining the concentration of UCH-L1 and/or GFAP in a test sample byan immunoassay as described herein, can be adapted for use in a varietyof automated and semi-automated systems (including those wherein thesolid phase comprises a microparticle), as described, e.g., U.S. Pat.No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881,2004/0018577, 2005/0054078, and 2006/0160164 and as commerciallymarketed e.g., by Abbott Laboratories (Abbott Park, Ill.) as AbbottPoint of Care (i-STAT® or i-STAT Alinity, Abbott Laboratories) as wellas those described in U.S. Pat. Nos. 5,089,424 and 5,006,309, and ascommercially marketed, e.g., by Abbott Laboratories (Abbott Park, Ill.)as ARCHITECT® or the series of Abbott Alinity devices.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., analyte antibody orcapture antibody) is attached (which can affect sandwich formation andanalyte reactivity), and the length and timing of the capture,detection, and/or any optional wash steps. Whereas a non-automatedformat such as an ELISA may require a relatively longer incubation timewith sample and capture reagent (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT® and any successor platform,Abbott Laboratories) may have a relatively shorter incubation time(e.g., approximately 18 minutes for ARCHITECT®). Similarly, whereas anon-automated format such as an ELISA may incubate a detection antibodysuch as the conjugate reagent for a relatively longer incubation time(e.g., about 2 hours), an automated or semi-automated format (e.g.,ARCHITECT® and any successor platform) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT® andany successor platform).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits, and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems. Asmentioned previously, the present disclosure is, for example, applicableto the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories)electrochemical immunoassay system that performs sandwich immunoassays.Immunosensors and their methods of manufacture and operation insingle-use test devices are described, for example in, U.S. Pat. No.5,063,081, U.S. Patent App. Publication Nos. 2003/0170881, 2004/0018577,2005/0054078, and 2006/0160164, which are incorporated in theirentireties by reference for their teachings regarding same.

In particular, with regard to the adaptation of an assay to the i-STAT®system, the following configuration is preferred. A microfabricatedsilicon chip is manufactured with a pair of gold amperometric workingelectrodes and a silver-silver chloride reference electrode. On one ofthe working electrodes, polystyrene beads (0.2 mm diameter) withimmobilized capture antibody are adhered to a polymer coating ofpatterned polyvinyl alcohol over the electrode. This chip is assembledinto an i-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the silicon chip, there is a specificbinding partner for UCH-L1 and/or GFAP, such as one or more UCH-L1and/or GFAP antibodies (one or more monoclonal/polyclonal antibody or afragment thereof, a variant thereof, or a fragment of a variant thereofthat can bind UCH-L1 and/or GFAP) or one or more anti-UCH-L1 and/or GFAPDVD-Igs (or a fragment thereof, a variant thereof, or a fragment of avariant thereof that can bind UCH-L1 and/or GFAP), either of which canbe detectably labeled. Within the fluid pouch of the cartridge is anaqueous reagent that includes p-aminophenol phosphate.

In operation, a sample from a subject suspected of suffering from TBI isadded to the holding chamber of the test cartridge, and the cartridge isinserted into the i-STAT® reader. A pump element within the cartridgepushes the sample into a conduit containing the chip. The sample isbrought into contact with the sensors allowing the enzyme conjugate todissolve into the sample. The sample is oscillated across the sensors topromote formation of the sandwich of approximately 2-12 minutes. In thepenultimate step of the assay, the sample is pushed into a waste chamberand wash fluid, containing a substrate for the alkaline phosphataseenzyme, is used to wash excess enzyme conjugate and sample off thesensor chip. In the final step of the assay, the alkaline phosphataselabel reacts with p-aminophenol phosphate to cleave the phosphate groupand permit the liberated p-aminophenol to be electrochemically oxidizedat the working electrode. Based on the measured current, the reader isable to calculate the amount of UCH-L1 and/or GFAP in the sample bymeans of an embedded algorithm and factory-determined calibration curve.

The automated and semi-automated systems described herein for use in themethods of the present disclosure can utilize one or more computerprograms, software or algorithms to provide the determination (readout)of whether to perform an imaging procedure (e.g., based on a positiveresult) or not to perform an imaging procedure (e.g., based on anegative result). For example, the computer program(s) or software(e.g., making use of an algorithm) can provide an interpretation(regardless of whether one, two or more samples are used) that: (1) whenthe level of GFAP and UCH-L1 is less than the reference level (orcutoff) that the result is negative meaning that no imaging procedurewill be performed; or (2) when the level or GFAP and/or UCH-L1 isgreater than or equal to the reference level (or cutoff) that the resultis positive meaning that an imaging procedure will be performed. Thecomputer program(s) or software can provide other appropriateinterpretations, such as, whether the reference level is or is notcorrelated with a positive head CT, the presence of an intracraniallesion or with control subjects that have not suffered a traumatic braininjury, whether the subject suffering from the TBI should be monitoredand/or treated with a TBI treatment, etc. Such computer programs orsoftware are well known in the art.

The methods and kits as described herein necessarily encompass otherreagents and methods for carrying out the immunoassay. For instance,encompassed are various buffers such as are known in the art and/orwhich can be readily prepared or optimized to be employed, e.g., forwashing, as a conjugate diluent, and/or as a calibrator diluent. Anexemplary conjugate diluent is ARCHITECT® conjugate diluent employed incertain kits (Abbott Laboratories, Abbott Park, Ill.) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.), which comprises a buffer containingMES, other salt, a protein blocker, and an antimicrobial agent.Additionally, as described in U.S. Patent Application No. 61/142,048filed Dec. 31, 2008, improved signal generation may be obtained, e.g.,in an i-STAT® cartridge format, using a nucleic acid sequence linked tothe signal antibody as a signal amplifier.

While certain embodiments herein are advantageous when employed toassess disease, such as traumatic brain injury, the assays and kits alsooptionally can be employed to assess UCH-L1 and/or GFAP in otherdiseases, disorders, and conditions as appropriate.

The method of assay also can be used to identify a compound thatameliorates diseases, such as traumatic brain injury. For example, acell that expresses UCH-L1 and/or GFAP can be contacted with a candidatecompound. The level of expression of UCH-L1 and/or GFAP in the cellcontacted with the compound can be compared to that in a control cellusing the method of assay described herein.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

13. Examples

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the presentdisclosure described herein are readily applicable and appreciable, andmay be made using suitable equivalents without departing from the scopeof the present disclosure or the aspects and embodiments disclosedherein. Having now described the present disclosure in detail, the samewill be more clearly understood by reference to the following examples,which are merely intended only to illustrate some aspects andembodiments of the disclosure, and should not be viewed as limiting tothe scope of the disclosure. The disclosures of all journal references,U.S. patents, and publications referred to herein are herebyincorporated by reference in their entireties.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 i-STAT® UCH-L1 Assay

The i-STAT® UCH-L1 assay was used in a TBI patient population study.Monoclonal antibody pairs, such as Antibody A as a capture monoclonalantibody and Antibody B and C as a detection monoclonal antibody, wereused. Antibody A is an exemplary anti-UCH-L1 antibody that wasinternally developed at Abbott Laboratories (Abbott Park, Ill.).Antibody B and C recognize different epitopes of UCH-L1 and enhance thedetection of antigen in the sample that were developed by BanyanBiomarkers (Alachua, Fla.). Other antibodies that were internallydeveloped at Abbott Laboratories (Abbott Park, Ill.) also show or areexpected to show similar enhancement of signal when used together ascapture antibodies or detection antibodies, in various combinations. TheUCH-L1 assay design was evaluated against key performance attributes.The cartridge configuration was Antibody Configuration: Antibody A(Capture Antibody)/Antibody B+C (Detection Antibody); Reagentconditions: 0.8% solids, 125 μg/mL Fab Alkaline Phosphatase clusterconjugate; and Sample Inlet Print: UCH-L1 standard. The assay time was10-15 min (with 7-12 min sample capture time).

Example 2 i-STAT® UCH-L1 Assay

The i-STAT® GFAP assay was used in a TBI patient population study.Monoclonal antibody pairs, such as Antibody A as a capture monoclonalantibody and Antibody B as a detection monoclonal antibody, were used.Antibody A and Antibody B are exemplary anti-GFAP antibodies that wereinternally developed at Abbott Laboratories (Abbott Park, Ill.). TheGFAP assay design was evaluated against key performance attributes. Thecartridge configuration was Antibody Configuration: Antibody A (CaptureAntibody)/Antibody B (Detection Antibody); Reagent conditions: 0.8%solids, 250 pg/mL Fab Alkaline Phosphatase cluster conjugate; and SampleInlet Print: GFAP specific. The assay time was 10-15 min (with 7-12 minsample capture time).

Example 3 TBI Population Study (TRACK-TBI)

The Transforming Research and Clinical Knowledge in Traumatic BrainInjury (TRACK-TBI) study is a large and complex project. Itsinstitutional and public-private partnership is comprised of over 11clinical sites, 7 Cores, for a total of nearly 50 collaboratinginstitutions, corporations, and philanthropy. An earlier TRACK-TBI Pilotstudy, based on clinical data from three clinical sites, helped refineTBI Common Data Elements and created a prototype of the TBI InformationCommons for the TRACK-TBI study.

Subject Groups:

A total of 2,700 to 3000 TBI patients were enrolled evenly across 3clinical groups, differentiated by clinical care path: 1. Patientsevaluated in the Emergency Department and discharged (ED); 2. Patientsadmitted to the hospital, but not to ICU (ADM); and 3. Patients admittedto the ICU (ICU). An additional 100 patients per clinical group (n=300)with extracranial trauma but no TBI were enrolled as controls for atotal enrollment of 3000 patients. This stratification plan facilitatedcomparative effectiveness research (CER) analysis and was notconstrained by traditional differentiation into “Mild/Moderate/Severe”TBI. Data collection was dependent on the clinical care path (ED, ADM,ICU) and requirements of each aim. Patients in each group werestratified into 3 cohorts that define the extent of data to becollected.

The controls were adult orthopedic trauma patients who met the followingcriteria: 1. An Abbreviated Injury Score of ≤4 (not life threatening)for their extremity and/or pelvis injury and/or rib fracture; 2. Met thesame inclusion and exclusion criteria as the TBI subjects except thatthe criterion of having undergone a CT or MRI in the ED for suspectedhead injury did not apply. TBI was ruled out for the current injury byinterviewing potential controls about loss of consciousness (LOC),disturbance of consciousness, and posttraumatic amnesia (PTA)/RA; 3.Each site was provided a plan for the number of controls to targetaccording to age and gender distributions derived from the TBI Cohort;and 4. Controls were enrolled into the CA-MRI cohort for follow-up anddrop to comprehensive assessment (CA) at 2-weeks if unable to completethe MRI visit.

Subject Eligibility:

Adult patients were enrolled of all ages presenting to the EmergencyDepartment (ED) with a history of acute TBI as per American Congress ofRehabilitation Medicine (ACRM) Criteria, in which the patient hadsustained a traumatically induced physiological disruption of brainfunction, as manifested by ≥one of the following: any period of loss ofconsciousness (LOC); any loss of memory for events (e.g., amnesia)immediately before or after the accident; any alteration of mental stateat the time of the accident (feeling dazed, disoriented, and/orconfused); and/or focal neurologic deficits that may or may not bepermanent. Traumatically induced included the head being struck, thehead striking an object, or the brain undergoing anacceleration/deceleration movement (e.g., whiplash) without directexternal trauma to the head.

The Inclusion/Exclusion Criteria used is shown in Table 2.

TABLE 2 Data Criterion Source Comments Inclusion Criteria 1. Age 0-100Chart 2. Documented/verified TBI (ACRM Chart, Criteria) Interview 3.Injury occurred <24 hours ago Chart, Interview 4. Acute brain CT forclinical care Chart Subject must have brain CT scan 5. Visualacuity/hearing adequate for Chart, testing Interview 6. Fluency inEnglish or Spanish Chart, Test battery or personnel availabilityInterview 7. Ability to provide informed consent Interview ExclusionCriteria 1. Significant polytrauma that would Chart Significant bodytrauma may confound interfere with follow-up and outcome TBI outcomestesting. assessment 2. Prisoners or patients in custody Chart, Interview3. Pregnancy in female subjects Chart, Interview 4. Patients onpsychiatric hold (e.g., Chart 5150, 5250) 5. Major debilitating baselinemental Chart, Debilitating psychiatric disorders can health disorders(e.g., schizophrenia Interview significantly impact the reliability ofor bipolar disorder) that would follow up and/or pose difficulties ininterfere with follow-up and the attributing to index TBI. validity ofoutcome assessment 6. Major debilitating neurological Chart, Documenteddebilitating baseline disease (e.g., stroke, CVA, dementia, Interviewcognitive impairment will confound tumor) impairing baseline awarenessoutcome assessment in addition to not cognition or validity of follow-upbeing fully consentable. and outcome assessment 7. Significant historyof pre-existing Chart, conditions that would interfere with Interviewfollow-up and outcome assessment (e.g., substance abuse, alcoholism,HIV/AIDS, major transmittable diseases that may interfere with consent,end-stage cancers, learning disabilities, developmental disorders) 8.Contraindications to MRI (for MRI CA + MRI cohort) Screening 9. Lowlikelihood of follow-up (e.g., Interview participant or familyindicating low interest, residence in another state or country,homelessness or lack of reliable contacts) 10. Current participant in anChart, Exception to co-enrollment exclusion is interventional trial(e.g., drug, device, Interview made for sites participating inbehavioral) Resuscitation Outcomes Consortium Prehospital TranexamicAcid for TBI Study. 11. Penetrating TBI Chart 12. Spinal cord injurywith ASIA score Chart of C or worse

For each of the 3 clinical groups (i.e., ED, ADM, and ICU), the subjectswere further placed into one of three different assessment cohorts:Brief Assessment (BA Cohort), Compressive Assessment (CA) Cohort, orComprehensive Assessment+MRI (CA+MRI) Cohort. See Table 3 for Milestoneplan with 80% follow up rate.

TABLE 3 Year 1 Year 2 CA + CA + Year 3 Year 4 Total Group MRI CA N MRICA N CA BA N BA N ED 150 87 237 50 58 108 155 100 255 300 900 ADM 150 87237 50 58 108 155 100 255 300 900 ICU 150 87 237 50 58 108 155 100 255300 900 Controls 0 99 99 0 66 66 135 0 135 0 300 Total 450 360 810 150240 390 600 300 900 900 3000

The Brief Assessment (BA) Cohort included 1200 total subjects, with 400subjects each for ED, ADM, and ICU Groups. The following data wasgathered for the BA Cohort: demographic and full clinical course data;blood draw for serum, plasma, DNA and RNA on Day 1 (<24 hours ofinjury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);clinical brain CT scan from Day 1 acquired as part of hospital course;and outcome data collected via structured telephone interview at 2weeks, 3, 6, and 12 months using NIH TBI-CDEs v.2.0 Core outcomemeasures as published on the NINDS CDE website.

The Compressive Assessment (CA) Cohort included 1200 total subjects,with 300 subjects+100 controls each for ED, ADM, and ICU Groups. Thefollowing data was gathered for the CA Cohort: demographic and fullclinical course data; high density daily clinical data for ADM and ICUGroups; blood draw for serum, plasma, RNA, and DNA on Day 1 (<24 hoursof injury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);blood draw for serum, plasma and RNA of Day 3 (48-72 hours) and 5(96-120 hours) for ADM and ICU; collection of cerebrospinal fluid ondays 1 through 5 (optional for sites to include this component); allclinical brain CT scans acquired as part of hospital course; blood drawfor serum, plasma and RNA at 2 weeks and 6 months; and outcome datacollected via structured in-person interview at 2 weeks, 6, and 12months and at 3 months via structured telephone interview using NIHTBI-CDEs v.2.0 Core, Basic and Supplemental outcome measures.

The Comprehensive Assessment+MRI (CA+MRI) Cohort included 600 totalsubjects, with 200 each for ED, ADM, and ICU Groups. The following datawas gathered for the CA+MRI Cohort: demographic and full clinical coursedata; high density daily clinical data for ADM and ICU Groups; blooddraw for serum, plasma, RNA, and DNA on Day 1 (<24 hours of injury);repeat blood draw for serum within 3-6 hours of the Day 1 baselinecollection (optional for sites to include this component); blood drawfor serum, plasma, and RNA on Day 3 (48-72 hours) and 5 (96-120 hours)for ADM and ICU; collection of cerebrospinal fluid on days 1 through 5(optional for sites to include this component); all clinical head CTscans acquired as part of hospital course; blood draw for serum, plasmaand RNA at 2 weeks and 6 months; 3T research MRI acquired at 2 weeks and6 months; and outcome data collected via structured in-person interviewat 2 weeks, 6, and 12 months and at 3 month via structured telephoneinterview using NIH TBI-CDEs v.2.0 Core, Basic, and Supplemental outcomemeasures.

Upon enrollment, data collection began in the hospital. For CA+MRIpatients, the 2-week MRI was completed at 14 days±4 days from the dateof injury. Corresponding 2-week outcomes were completed ±3 days of the2-week MRI. For CA and BA patients, 2-week outcomes were completed ±4days of 14 days from the date of injury. Outcomes at 3 months werecompleted ±7 days of 90 days from the date of injury. For CA+MRIpatients, MRIs at 6 months were completed ±14 days of 180 days from thedate of injury, with corresponding 6-month outcomes ±14 days of the6-month MRI. For CA and BA patients, 6-month outcomes were completed ±14days of 180 days from the date of injury. BTACT should be completed with±7 days of Outcomes (but not on the same day and no greater than 201days from injury). Outcomes at 12 months were completed ±30 days of 360days from the date of injury.

UCH-L1 and GFAP were measured in a small sample size of 59 TRACK TBIpatients in the i-STAT assay format (Table 4).

TABLE 4 Subject Characteristics by CT Scan and MRI Result Subject TotalCT or MRI Positive* CT or MRI Negative* Characteristics (n = 59) (n =46, 77.97%) (n = 13, 22.03%) P value Age 40.0 [24.0 to 00.0] 45.5 [23.0to 00.0] 50.0 [39.0 to 57.0] 0.7419 Sex Male 50/59 (85%) 39/46 (85%)11/13 (85%) 1.0000 Female 9/59 (15%) 7/46 (15%) 2/13 (15%)Race/Ethnicity African-American or African 6/58 (10%) 4/45 (9%) 2/13(15%) 0.2398 Caucasian 48/58 (83%) 39/45 (87%) 9/13 (69%) Hispanic 4/58(7%) 2/45 (4%) 2/13 (15%) TBI History Yes, with No LOC 9/56 (16%) 3/43(7%) 6/13 (46%) 0.0037 Yes, with LOC 8/56 (14%) 6/43 (14%) 2/13 (15%) NoPrior TBI 39/56 (70%) 34/43 (79%) 5/13 (38%) ED Presentation Loss ofConsciousness No 6/58 (10%) 2/45 (4%) 4/13 (31%) 0.0227 Yes 47/58 (81%)38/45 (84%) 9/13 (69%) Unknown 5/58 (9%) 5/45 (11%) Glasgow Coma Scale15.0 [3.0 to 15.0] 14.0 [3.0 to 15.0] 15.0 [15.0 to 15.0] 0.0162 GlasgowComa Scale Classification Severe (3-8) 16/59 (27%) 16/46 (35%) 0.0177Moderate (9-12) 3/59 (5%) 3/46 (7%) Mild (13-15) 40/59 (68%) 27/46 (59%)13/13 (100%) Mechanism of Injury Motor vehicle (driver/passenger) 10/59(17%) 9/46 (20%) 1/13 (8%) 0.2975 Motorcycle/ATV/golf cart 5/59 (8%)3/46 (7%) 2/13 (15%) (driver/passenger) Individual struck by any type of3/59 (5%) 2/46 (4%) 1/13 (8%) vehicle Fall from a moving object 3/59(5%) 3/46 (7%) (bike/skateboard/horse/etc.) Fall from stationary object27/59 (46%) 20/46 (43%) 7/13 (54%) (roof/ladder/etc.) Assault 10/59(17%) 9/46 (20%) 1/13 (8%) Struck on head by object, not 1/59 (2%) 1/13(8%) assault (tree/etc.) Alcohol Level (g/dL) 0.1 [0.0 to 0.2] 0.1 [0.0to 0.2] 0.0 [0.0 to 0.0] 0.1588 Drug Screen Negative 51/59 (86%) 41/46(89%) 10/13 (77%) 0.3567 Positive 8/59 (14%) 5/46 (11%) 3/13 (23%)Biomarker Results Collection Time Since Injury 771.0 (+/−339.8) 779.4(+/−296.8) 743.0 (+/−468.7) 0.7383 (Minutes) GFAP (pg/mL) 643.8 [188.6to 2138.6] 876.6 [519.7 to 2409.5] 31.3 [26.3 to 166.2] <0.0001 UCH-L1(pg/mL) 342.5 [102.8 to 718.3] 514.0 [167.2 to 859.8] 62.4 [44.5 to136.8] <0.0001 Prognostic Scores Glasgow Outcome Scale (3 months) 6.0[5.0 to 7.0] 5.5 [4.0 to 7.0] 7.0 [7.0 to 7.0] 0.0130 Glasgow OutcomeScale (6 months) 6.0 [5.0 to 7.0] 6.0 [4.0 to 7.0] 7.0 [5.5 to 7.5]0.1941 Glasgow Outcome Scale (12 months) 7.0 [5.0 to 8.0] 6.5 [5.0 to8.0] 7.0 [6.0 to 8.0] 0.4412 Rivermead Questionnaire First 0.0 [0.0 to2.0] 0.0 [0.0 to 2.5] 0.0 [0.0 to 2.0] 0.8378 3 Items (6 months)Rivermead Questionnaire Last 9.0 [4.0 to 15.0] 8.5 [4.0 to 15.0] 13.0[0.0 to 27.0] 0.5449 13 Items (6 months) WAIS-III Processing Speed Index30.0 [5.0 to 55.0] 30.0 [5.0 to 50.0] 43.0 [18.0 to 77.0] 0.3235 (6months) Satisfaction with Life Scale 21.5 (+/−6.2) 21.7 (+/−5.7) 20.4(+/−8.5) 0.6205 (6 months) Functional Independence Measure 126.0 [125.0to 126.0] 126.0 [124.0 to 126.0] 126.0 [126.0 to 126.0] 0.2958 (6months) *24 subjects received an MRI Continuous variables are presentedas median [25-75% Inter Quartile Range] and compared using Wilcoxon ranksum test or Mean (+/−SD) and compared using a t-test based on thedistribution of the data. Categorical variables are presented asnumber/total (percent) and compared using Chi-Square or Fisher's exacttest.

In addition to a blood draw within 24 hours of brain injury, eachpatient had an extensive medical evaluation including head CT,neuropsychiatric testing, Glasgow Coma Score (GCS), and many patientsalso had a follow up MRI within 2 weeks of injury. Following ameticulous standardized blood draw protocol and processing, plasmasamples were aliquoted for storage at −80° C., later thawed and tested.Each sample was run in duplicate with the listed results being anaverage of the two runs. FIGS. 1A and 1B show that UCH-L1 levels (FIG.1A) and GFAP levels (FIG. 1B) correlated with injury throughout thefirst 24 hours after injury (range approximately 2-23 hours) in anexemplary subset of subjects. Table 4 shows that ethanol (ETOH) levelsdid not correlate with biomarker levels (Pearson Correlation=0.023,p-value=0.89) as ETOH consumption is frequently related to TBI, inparticular severe TBI.

Table 5 shows the results of using UCH-L1 and GFAP cut-off levels of 100pg/mL and 300 pg/mL compared to imaging results.

TABLE 5 Biomarker Cut-offs (Reference Levels) for UCH-L1 and GFAPCompared to Imaging Results Biomarker Biomarker Imaging Cutoff ImagingCombination Sensitivity Specificity PPV NPV Method (pg/mL) MethodPositive Negative (95% CI) (95% CI) (95% CI) (95% CI) CT 100 Positive 4411  100.00% (44/44) 26.67% (4/15) 80.00% (44/55) 100.00% (4/4) (A) (B)(91.96%, 100.00%) (7.79%, 55.10%) (67.03%, 89.57%) (39.76%, 100.00%)Negative  0 4 (C) (D) MRI or CT 100 Positive 46 9 100.00% (46/46) 30.77%(4/13) 83.64% (46/55) 100.00% (4/4) (A) (B) (92.29%, 100.00%) (9.09%,61.43%) (71.20%, 92.23%) (39.76%, 100.00%) Negative  0 4 (C) (D) CT 300Positive 41 2 93.18% (41/44) 86.67% (13/15) 95.35% (41/43) 81.25%(13/16) (A) (B) (81.34%, 98.57%) (59.54%, 98.34%) (84.19%, 99.43%)(54.35%, 95.95%) Negative  3 13  (C) (D) MRI or CT 300 Positive 42 191.30% (42/46) 92.31% (12/13) 97.67% (42/43) 75.00% (12/16) (A) (B)(79.21%, 97.58%) (63.97%, 99.81%) (87.71%, 99.94%) (47.62%, 92.73%)Negative  4 12  (C) (D) Sensitivity = A/(A + C) times 100 Specificity =D/(B + D) times 100 PPV = A/(A + B) times 100 NPV = D/(C + D) times 100*Note: In the above table, “Biomarker Cutoff” and Biomarker ReferenceLevel are used interchangeably herein

MRI.

FIGS. 2A and 2B show receiver operating characteristic (ROC) analysis ofUCH-L1 levels (FIG. 2A; AUC of 0.815) and GFAP levels (FIG. 2B; AUC0.835) correlated with the presence or absence of intracranial lesionresult as detected using MRI for all of the subjects by Time Point. FIG.3A shows the ROC analysis of the absolute amount of UCH-L1 levels(“absolute delta”) (i.e., change between the UCH-L1 levels in the firsttime point (“Time Point 1”) to the UCH-L1 levels in the second timepoint (“Time Point 2”)) correlated with the presence or absence ofintracranial lesion result as detected using MRI (AUC=0.715). FIG. 3Bshows the ROC analysis of the absolute amount of GFAP levels (“absolutedelta”) (i.e., change between the GFAP levels in the first time point(“Time Point 1”) to the GFAP levels in the second time point (“TimePoint 2”)) correlated with the presence or absence of intracraniallesion result as detected using MRI (AUC=0.592). The sensitivity andspecificity of reference levels based on the ROC curves are shown inTables 6 and 7.

TABLE 6 Biomarker Levels (pg/mL) Sensitivity Specificity UCH-L1 88 83.2430.37 97 80.35 33.25 117 75.43 40.31 GFAP 24 94.8 30.89 44 91.04 40.0557 90.17 45.55 79 87.57 50 115 85.26 60.73 185 80.06 70.68

TABLE 7 Absolute Amount Biomarker (pg/mL) Sensitivity Specificity UCH-L135 81.75 31.25 48 77.78 37.5 62 73.02 40.63 GFAP 18 94.44 31.25 34 91.2740.63 60 88.89 50 135 80.16 62.5

CT Scan.

FIGS. 4A and 4B show ROC analysis of UCH-L1 levels (FIG. 4A) and GFAPlevels (FIG. 4B) correlated with the presence or absence of intracraniallesion result as detected using CT scan for all of the subjects by TimePoint. FIG. 5A shows the ROC analysis of the absolute amount of UCH-L1levels (“absolute delta”) (i.e., change between the UCH-L1 levels in thefirst time point (“Time Point 1”) to the UCH-L1 levels in the secondtime point (“Time Point 2”)) correlated with the presence or absence ofintracranial lesion result as detected using CT scan. FIG. 5B shows theROC analysis of the absolute amount of GFAP levels (“absolute delta”)(i.e., change between the GFAP levels in the first time point (“TimePoint 1”) to the GFAP levels in the second time point (“Time Point 2”))correlated with the presence or absence of intracranial lesion result asdetected using CT scan.

MRI or CT Scan.

FIGS. 6A and 6B show ROC analysis of UCH-L1 levels (FIG. 6A; AUC=0.683)and GFAP levels (FIG. 6B; AUC=0.847) correlated with the presence orabsence of intracranial lesion result as detected using MRI or CT scanfor all of the subjects by Time Point. FIG. 7A shows the ROC analysis ofthe absolute amount of UCH-L1 levels (“absolute delta”) (i.e., changebetween the UCH-L1 levels in the first time point (“Time Point 1”) tothe UCH-L1 levels in the second time point (“Time Point 2”)) correlatedwith the presence or absence of intracranial lesion result as detectedusing MRI or CT scan (AUC=0.668). FIG. 7B shows the ROC analysis of theabsolute amount of GFAP levels (“absolute delta”) (i.e., change betweenthe GFAP levels in the first time point (“Time Point 1”) to the GFAPlevels in the second time point (“Time Point 2”)) correlated with thepresence or absence of intracranial lesion result as detected using MRIor CT scan (AUC=0.799). The sensitivity and specificity of referencelevels based on the ROC curves are shown in Tables 8 and 9.

TABLE 8 Biomarker Levels (pg/mL) Sensitivity Specificity UCH-L1 87 84.0730.77 96 80.16 33.24 GFAP 22 95.04 30.77 40 91.65 40.11 63 89.56 50 10085.64 60.17 156 81.98 70.06

TABLE 9 Absolute Amount Biomarker (pg/mL) Sensitivity Specificity UCH-L146 76.98 31.25 66 70.5 42.19 GFAP 11 96.4 31.25 21 93.53 40.63 42 89.2150 90 84.17 60.94 124 80.58 65.63

MRI but Negative CT Scan.

FIGS. 8A and 8B show ROC analysis of UCH-L1 levels (FIG. 8A; AUC of0.620) and GFAP levels (FIG. 8B; AUC of 0.768) correlated with a presentintracranial lesion result using MRI but negative CT scan by time point.The sample is taken within 24 hours of head injury. FIG. 9A shows areceiver operating characteristic (ROC) analysis of absolute amount(“absolute delta”) of UCH-L1 levels (i.e., the absolute differencebetween UCH-L1 levels at Time Point 2 and UCH-L1 levels at Time Point 1)correlated with a present intracranial lesion result using MRI butnegative CT scan (AUC=0.626). The sample at Time Point 1 is taken within24 hours of head injury while the sample at Time Point 2 is taken about3 to about 6 hours after the Time Point 1 sample is taken. FIG. 9B showsa receiver operating characteristic (ROC) analysis of absolute amount(“absolute delta”) of GFAP levels (i.e., the absolute difference betweenGFAP levels at Time Point 2 and GFAP levels at Time Point 1) correlatedwith a present intracranial lesion result using MRI but negative CT scan(AUC=0.775). The sample at Time Point 1 is taken within 24 hours of headinjury while the sample at Time Point 2 is taken about 3 to about 6hours after the Time Point 1 sample is taken. The sensitivity andspecificity of reference levels based on the ROC curves are shown inTables 10 and 11.

TABLE 10 Biomarker Levels (pg/mL) Sensitivity Specificity UCH-L1 8484.06 30.03 93 80.44 33.23 114 76.09 40.26 GFAP 22 89.86 30.35 42 84.7840.58 66 83.33 50.48 81 81.16 53.67

TABLE 11 Absolute Amount Biomarker (pg/mL) Sensitivity SpecificityUCH-L1 46 83.33 31.58 61 80.56 36.84 82 75 47.37 GFAP 14 91.67 31.58 2191.67 42.11 34 86.11 49.12 62 80.56 57.9

The combination of GFAP and UCH-L1 was analyzed in CT negative patientsthat had positive MRI intracranial lesion results. The sensitivity andspecificity of the GFAP and UCH-L1 reference levels are shown in Table12.

TABLE 12 GFAP or UCH-L1 (Equal to or greater than Cutoff) GFAP Cutoff(pg/mL) UCH-L1 Cutoff (pg/mL) Sensitivity Specificity 22 84 97.83 14.4322 93 97.1 15.74 22 114 97.1 19.02 22 180 94.2 24.26 22 250 92.75 26.2322 350 91.3 28.52 42 84 96.38 18.36 42 93 95.65 20.33 42 114 96.65 23.9342 180 91.3 30.16 42 250 88.41 33.44 42 350 86.96 37.38 50 180 90.5833.44

The combination of GFAP and UCH-L1 was analyzed in CT negative patientsthat had negative MRI intracranial lesion results. The sensitivity andspecificity of the GFAP and UCH-L1 reference levels are shown in Table13.

TABLE 13 GFAP or UCH-L1 (Equal to or greater than Cutoff) GFAP Cutoff(pg/mL) UCH-L1 Cutoff (pg/mL) Sensitivity Specificity 450 400 57.2580.66 450 500 52.9 83.61 450 600 52.17 87.21 450 700 49.28 87.54 650 40044.93 84.26 650 500 40.58 87.54 650 600 39.86 91.15 650 700 36.96 91.48

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A method of aiding in the determination of whether to performan imaging procedure on a human subject that has sustained or may havesustained an injury to the head, the method comprising: a) performing anassay on a sample obtained from the subject within about 24 hours aftera suspected injury to the head to measure or detect a level of an earlybiomarker in the sample, said early biomarker comprising ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof, in the sample; and b) performing animaging procedure on the subject when the level of the early biomarkerin the sample is higher than a reference level of the early biomarkerand not performing an imaging procedure on the subject when the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker, wherein the reference level is (a) determined by anassay having a sensitivity of between at least about 70% to 100% and aspecificity of between at least about 30% to 100%; or (b) between atleast about 20 pg/mL to about 200 pg/mL.

Clause 2. The method of clause 1, wherein the reference level isdetermined by an assay having a sensitivity of at least about 80% and aspecificity of at least about 30%.

Clause 3. The method of clause 1, wherein the reference level is betweenat least about 80 pg/mL to about 150 pg/mL.

Clause 4. The method of any one of clauses 1 to 3, wherein the subjecthas received an imaging procedure before or after the assay isperformed.

Clause 5. The method of clause 4, wherein the subject is suspected ofhaving a traumatic brain injury based on the imaging procedure.

Clause 6. The method of any one of clauses 1 to 5, wherein the imagingprocedure is a magnetic resonance imaging or a head computerizedtomography (CT) scan.

Clause 7. The method of clause 6, wherein the reference level iscorrelated with a positive MRI scan or positive CT scan.

Clause 8. The method of clause 7, wherein the reference level iscorrelated with the presence of an intracranial lesion.

Clause 9. The method of clause 1, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 10. The method of any one of clauses 1 to 9, wherein thereference level for GFAP is between about 20 pg/mL and about 200 pg/mL.

Clause 11. The method of any one of clauses 1 to 10, wherein thereference level for UCH-L1 is about 80 pg/mL and about 150 pg/mL.

Clause 12. The method of any one of clauses 1 to 11, wherein the sampleis taken within about 0 to about 12 hours after the suspected injury tothe head.

Clause 13. A method of aiding in the determination of whether to performan imaging procedure on a human subject that has sustained or may havesustained an injury to the head, the method comprising: a) performing anassay on at least two samples obtained from the subject, the firstsample taken from the subject within 24 hours of a suspected injury andthe second sample taken from the subject from about 3 to about 6 hoursafter the first sample is taken; b) detecting in the at least twosamples an early biomarker of traumatic brain injury, said earlybiomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof; and c)performing an imaging procedure on the subject when the level of theearly biomarker decreases or increases by at least an absolute amountfrom the first sample to the second sample and not performing an imagingprocedure on the subject when there is no decrease or increase by atleast an absolute amount in the level of the early biomarker from thefirst sample to the second sample.

Clause 14. The method of clause 13, wherein the subject has received animaging procedure before or after the assay is performed.

Clause 15. The method of clause 14, wherein the subject is suspected ashaving a traumatic brain injury based on the imaging procedure.

Clause 16. The method of any one of clauses 13 to 15, wherein theimaging procedure is a magnetic resonance imaging or a head computerizedtomography (CT) scan.

Clause 17. The method of clause 16, wherein the absolute amount iscorrelated with a positive MRI scan or positive CT scan.

Clause 18. The method of clause 17, wherein the absolute amount iscorrelated with the presence of an intracranial lesion.

Clause 19. The method of clause 18, wherein the absolute amount isdetermined by an assay having a sensitivity of between at least about70% to 100% and a specificity of between at least about 30% to 100%.

Clause 20. The method of clause 19, wherein the absolute amount isdetermined by an assay having (a) a sensitivity of at least about 80%and a specificity of at least about 30%; or (b) a sensitivity of atleast about 75% and a specificity of at least about 40%.

Clause 21. The method of clause 19 or 20, wherein the absolute amount isbetween at least about 10 pg/mL and at least about 150 pg/mL.

Clause 22. The method of clause 23, wherein the early biomarker isUCH-L1 and the absolute amount is between at least about 30 pg/mL toabout 100 pg/mL, the early biomarker is GFAP and the absolute amount isbetween at least about 10 pg/mL to about 150 pg/mL, or a combinationthereof.

Clause 23. The method of any one of clauses 13 to 22, wherein the firstsample is taken within about 0 to about 12 hours after the suspectedinjury to the head.

Clause 24. The method of any one of clauses 13 to 23, wherein the secondsample is taken from the subject between about 3 hours to about 6 afterthe first sample.

Clause 25. The method of any one of clauses 1 to 24, wherein measuringthe level of UCH-L1 is done by an immunoassay or clinical chemistryassay.

Clause 26. The method of any one of clauses 1 to 25, wherein measuringthe level of UCH-L1 comprises: A. contacting the sample, eithersimultaneously or sequentially, in any order with: (1) a UCH-L1-captureantibody, which binds to an epitope on UCH-L1 or UCH-L1 fragment to forma UCH-L1-capture antibody-UCH-L1 antigen complex, and (2) aUCH-L1-detection antibody which includes a detectable label and binds toan epitope on UCH-L1 that is not bound by the UCH-L1-capture antibody,to form a UCH-L1 antigen-UCH-L1-detection antibody complex, such that aUCH-L1-capture antibody-UCH-L1 antigen-UCH-L1-detection antibody complexis formed, and B. measuring the amount or concentration of UCH-L1 in thesample based on the signal generated by the detectable label in theUCH-L1-capture antibody-UCH-L1 antigen-UCH-L1-detection antibodycomplex.

Clause 27. The method of any one of clauses 1 to 26, wherein measuringthe level of GFAP is done by an immunoassay or clinical chemistry assay.

Clause 28. The method of any one of clauses 1 to 27, wherein measuringthe level of GFAP comprises: A. contacting the sample, eithersimultaneously or sequentially, in any order with: (1) a GFAP-captureantibody, which binds to an epitope on GFAP or GFAP fragment to form aGFAP-capture antibody-GFAP antigen complex, and (2) a GFAP-detectionantibody which includes a detectable label and binds to an epitope onGFAP that is not bound by the GFAP-capture antibody, to form a GFAPantigen-GFAP-detection antibody complex, such that a GFAP-captureantibody-GFAP antigen-GFAP-detection antibody complex is formed, and B.measuring the amount or concentration of GFAP in the sample based on thesignal generated by the detectable label in the GFAP-captureantibody-GFAP antigen-GFAP-detection antibody complex.

Clause 29. The method of any one of clauses 1 to 28, wherein the sampleis selected from the group consisting of a whole blood sample, a serumsample, a cerebrospinal fluid sample, and a plasma sample.

Clause 30. The method of any one of clauses 1 to 29, wherein the sampleis obtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

Clause 31. The method of any one of clauses 1 to 30, wherein the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 32. The method of clause 31, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 33. The method of any one of clauses 1 to 29, wherein the sampleis obtained from a subject that suffers from an autoimmune disease, ametabolic disorder, a brain tumor, hypoxia, a virus, meningitis,hydrocephalus or combinations thereof.

Clause 34. The method of any one of clauses 1 to 33, wherein said methodcan be carried out on any subject without regard to factors selectedfrom the group consisting of the subject's clinical condition, thesubject's laboratory values, the subject's classification as sufferingfrom mild, moderate, severe, or moderate to severe traumatic braininjury, the subject's exhibition of low or high levels of UCH-L1, andthe timing of any event wherein said subject may have sustained aninjury to the head.

Clause 35. The method of any one of clauses 1 to 34, further comprisingtreating the subject with a mild traumatic brain injury treatment.

Clause 36. The method of any one of clauses 1 to 34, further comprisingmonitoring the subject.

Clause 37. The method of any one of clauses 1 to 36, wherein the sampleis a whole blood sample.

Clause 38. The method of any one of clauses 1 to 36, wherein the sampleis a serum sample.

Clause 39. The method of any one of clauses 1 to 36, wherein the sampleis a plasma sample.

Clause 40. The method of any one of clauses 37 to 39, wherein the assayis an immunoassay.

Clause 41. The method of any one of clauses 37 to 39, wherein the assayis a clinical chemistry assay.

Clause 42. The method of any one of clauses 37 to 39, wherein the assayis a single molecule detection assay.

Clause 43. A method of aiding in the determination of whether to performan imaging procedure on a human subject that has sustained or may havesustained an injury to the head, the method comprising:

-   -   a) performing an assay on a sample obtained from the subject        within about 24 hours after a suspected injury to the head to        measure or detect a level of an early biomarker in the sample,        said early biomarker comprising ubiquitin carboxy-terminal        hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP),        or a combination thereof, in the sample; and    -   b) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker in the sample is        higher than a reference level of the early biomarker and not        performing an imaging procedure on the subject when the level of        the early biomarker in the sample is lower than a reference        level of the early biomarker,        wherein the reference level is (a) determined by an assay having        a sensitivity of between at least about 70% to 100% and a        specificity of between at least about 30% to 100%; or (b)        between at least about 20 pg/mL to about 200 pg/mL.

Clause 44. A method of evaluating a human subject to determine whetherto perform an imaging procedure for a head injury, the methodcomprising:

-   -   a) performing an assay on a sample obtained from the subject        within about 24 hours after a suspected injury to the head to        measure a level of an early biomarker in the sample, said early        biomarker comprising ubiquitin carboxy-terminal hydrolase L1        (UCH-L1), glial fibrillary acidic protein (GFAP), or a        combination thereof, in the sample; and    -   b) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker in the sample is        higher than a reference level of the early biomarker and not        performing an imaging procedure on the subject when the level of        the early biomarker in the sample is lower than a reference        level of the early biomarker,        wherein the reference level is (a) determined by an assay having        a sensitivity of between at least about 70% to 100% and a        specificity of between at least about 30% to 100%; or (b)        between at least about 20 pg/mL to about 200 pg/mL.

Clause 45. The method of clause 43 or 44, wherein the reference level isdetermined by an assay having a sensitivity of at least about 80% and aspecificity of at least about 30%.

Clause 46. The method of clause 43 or 44, wherein the reference level isbetween at least about 80 pg/mL to about 150 pg/mL.

Clause 47. The method of any one of clauses 43 to 46, wherein thesubject has received an imaging procedure before or after the assay isperformed.

Clause 48. The method of clause 47, wherein the subject is suspected ofhaving a traumatic brain injury based on the imaging procedure.

Clause 49. The method of any one of clause 43 to 48, wherein the imagingprocedure is a magnetic resonance imaging (MRI) or a head computerizedtomography (CT) scan.

Clause 50. The method of clause 49, wherein the reference level iscorrelated with a positive MRI scan or positive CT scan.

Clause 51. The method of clause 50, wherein the reference level iscorrelated with the presence of an intracranial lesion.

Clause 52. The method of clause 43 or 44, wherein the reference level iscorrelated with control subjects that have not sustained a head injury.

Clause 53. The method of any one of clause 43 to 52, wherein thereference level for GFAP is between about 20 pg/mL and about 200 pg/mL.

Clause 54. The method of any one of clauses 43 to 53, wherein thereference level for UCH-L1 is about 80 pg/mL and about 150 pg/mL.

Clause 55. The method of any one of clauses 43 to 54, wherein the sampleis taken within about 0 to about 12 hours after the suspected injury tothe head.

Clause 56. A method of aiding in the determination of whether to performan imaging procedure on a human subject that has sustained or may havesustained an injury to the head, the method comprising:

-   -   a) performing an assay on at least two samples obtained from the        subject, the first sample taken from the subject within 24 hours        of a suspected injury and the second sample taken from the        subject from about 3 to about 6 hours after the first sample is        taken;    -   b) detecting in the at least two samples an early biomarker of        traumatic brain injury, said early biomarker comprising        ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial        fibrillary acidic protein (GFAP), or a combination thereof; and    -   c) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker decreases or        increases by at least an absolute amount from the first sample        to the second sample and not performing an imaging procedure on        the subject when there is no decrease or increase by at least an        absolute amount in the level of the early biomarker from the        first sample to the second sample.

Clause 57. A method of evaluating a human subject to determine whetherto perform an imaging procedure for a head injury, the methodcomprising:

-   -   a) performing an assay on at least two samples obtained from the        subject, the first sample taken from the subject within 24 hours        of a suspected injury and the second sample taken from the        subject from about 3 to about 6 hours after the first sample is        taken;    -   b) detecting in the at least two samples an early biomarker of        traumatic brain injury, said early biomarker comprising        ubiquitin carboxy-terminal hydrolase L1 (UCH-L 1), glial        fibrillary acidic protein (GFAP), or a combination thereof; and    -   c) determining whether to perform an imaging procedure on the        subject when the level of the early biomarker decreases or        increases by at least an absolute amount from the first sample        to the second sample and not performing an imaging procedure on        the subject when there is no decrease or increase by at least an        absolute amount in the level of the early biomarker from the        first sample to the second sample.

Clause 58. The method of clause 56 or 57, wherein the subject hasreceived an imaging procedure before or after the assay is performed.

Clause 59. The method of clause 58, wherein the subject is suspected ashaving a traumatic brain injury based on the imaging procedure.

Clause 60. The method of any one of clauses 56 to 59, wherein theimaging procedure is a magnetic resonance imaging or a head computerizedtomography (CT) scan.

Clause 61. The method of clause 60, wherein the absolute amount iscorrelated with a positive MRI scan or positive CT scan.

Clause 62. The method of clause 61, wherein the absolute amount iscorrelated with the presence of an intracranial lesion.

Clause 63. The method of clause 62, wherein the absolute amount isdetermined by an assay having a sensitivity of between at least about70% to 100% and a specificity of between at least about 30% to 100%.

Clause 64. The method of clause 63, wherein the absolute amount isdetermined by an assay having (a) a sensitivity of at least about 80%and a specificity of at least about 30%; or (b) a sensitivity of atleast about 75% and a specificity of at least about 40%.

Clause 65. The method of clause 63 or 64, wherein the absolute amount isbetween at least about 10 pg/mL and at least about 150 pg/mL.

Clause 66. The method of clause 65, wherein the early biomarker isUCH-L1 and the absolute amount is between at least about 30 pg/mL toabout 100 pg/mL, the early biomarker is GFAP and the absolute amount isbetween at least about 10 pg/mL to about 150 pg/mL, or a combinationthereof.

Clause 67. The method of any one of clauses 56 to 66, wherein the firstsample is taken within about 0 to about 12 hours after the suspectedinjury to the head.

Clause 68. The method of any one of clauses 56 to 67, wherein the secondsample is taken from the subject between about 3 hours to about 6 afterthe first sample.

Clause 69. The method of any one of clauses 43 to 68, wherein measuringthe level of UCH-L1 is done by an immunoassay or clinical chemistryassay.

Clause 70. The method of any one of clauses 43 to 69, wherein measuringthe level of UCH-L1 comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a UCH-L1-capture antibody, which binds to an epitope on            UCH-L1 or UCH-L1 fragment to form a UCH-L1-capture            antibody-UCH-L1 antigen complex, and        -   (2) a UCH-L1-detection antibody which includes a detectable            label and binds to an epitope on UCH-L1 that is not bound by            the UCH-L1-capture antibody, to form a UCH-L1            antigen-UCH-L1-detection antibody complex,        -   such that a UCH-L1-capture antibody-UCH-L1            antigen-UCH-L1-detection antibody complex is formed, and    -   B. measuring the amount or concentration of UCH-L1 in the sample        based on the signal generated by the detectable label in the        UCH-L1-capture antibody-UCH-L1 antigen-UCH-L1-detection antibody        complex.

Clause 71. The method of any one of clauses 43 to 70, wherein measuringthe level of GFAP is done by an immunoassay or clinical chemistry assay.

Clause 72. The method of any one of clauses 43 to 71, wherein measuringthe level of GFAP comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a GFAP-capture antibody, which binds to an epitope on            GFAP or GFAP fragment to form a GFAP-capture antibody-GFAP            antigen complex, and        -   (2) a GFAP-detection antibody which includes a detectable            label and binds to an epitope on GFAP that is not bound by            the GFAP-capture antibody, to form a GFAP            antigen-GFAP-detection antibody complex,        -   such that a GFAP-capture antibody-GFAP            antigen-GFAP-detection antibody complex is formed, and    -   B. measuring the amount or concentration of GFAP in the sample        based on the signal generated by the detectable label in the        GFAP-capture antibody-GFAP antigen-GFAP-detection antibody        complex.

Clause 73. The method of any one of clauses 43 to 72, wherein the sampleis selected from the group consisting of a whole blood sample, a serumsample, a cerebrospinal fluid sample, and a plasma sample.

Clause 74. The method of any one of clauses 43 to 73, wherein the sampleis obtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

Clause 75. The method of any one of clauses 43 to 74, wherein the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 76. The method of clause 75, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 77. The method of any one of clauses 43 to 73, wherein the sampleis obtained from a subject that suffers from an autoimmune disease, ametabolic disorder, a brain tumor, hypoxia, a virus, meningitis,hydrocephalus or combinations thereof.

Clause 78. The method of any one of clauses 43 to 77, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of UCH-L1,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 79. The method of any one of clauses 43 to 76, further comprisingtreating the subject with a mild traumatic brain injury treatment.

Clause 80. The method of any one of clauses 43 to 76, further comprisingmonitoring the subject.

Clause 81. The method of any one of clauses 43 to 80, wherein the sampleis a whole blood sample.

Clause 82. The method of any one of clauses 43 to 80, wherein the sampleis a serum sample.

Clause 83. The method of any one of clauses 43 to 80, wherein the sampleis a plasma sample.

Clause 84. The method of any one of clauses 81 to 83, wherein the assayis an immunoassay.

Clause 85. The method of any one of clauses 81 to 83, wherein the assayis a clinical chemistry assay.

Clause 86. The method of any one of clauses 81 to 83, wherein the assayis a single molecule detection assay.

Clause 87. The method of any one of clauses 1 to 86, wherein the methodis adapted for use in an automated or semi-automated system.

Clause 88. The method of clause 87, wherein the automated orsemi-automated system utilizes a computer program to determine whetherto perform an imaging procedure on the subject or not to perform animaging procedure on the subject.

Clause 89. The method of clause 88, wherein the determination by thecomputer program that an imaging procedure needs to be performed isshown as a positive result.

Clause 90. The method of claim 88, wherein the determination by thecomputer program that an imaging procedure does not need to be performedis shown as a negative result.

What is claimed is:
 1. A method comprising: a) performing at least oneassay for at least one early biomarker selected from the groupconsisting of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glialfibrillary acidic protein (GFAP), and ubiquitin carboxy-terminalhydrolase L1 and glial fibrillary acidic protein on at least one samplethat is whole blood, serum, plasma, or cerebrospinal fluid obtained froma human subject within about 24 hours after an actual or suspectedinjury to the head; and b) performing a magnetic imaging (MRI) procedureon the subject and treating the subject for a moderate, severe, or amoderate to severe TBI when the level of UCH-L1, GFAP, or UCH-L1 andGFAP in the sample is higher than a reference level of UCH-L1, GFAP, orUCH-L1 and GFAP; and wherein the reference level is between at leastabout 20 pg/mL to about 200 pg/mL.
 2. The method of claim 1, wherein theassay is selected from the group consisting of an immunoassay, and aclinical chemistry assay.
 3. The method of claim 1, wherein the assay isperformed using single molecule detection or a point-of-care assay. 4.The method of claim 1, wherein the assay has a sensitivity of at leastabout 80% and a specificity of at least about 30%.
 5. The method ofclaim 1, wherein (a) the at least one early biomarker is UCH-L1 and thereference level for UCH-L1 is between at least about 80 pg/mL and about150 pg/mL, (b) the early biomarker is GFAP and the reference level forGFAP is between about 20 pg/mL and about 200 pg/mL, or (c) the earlybiomarker is UCH-L1 and GFAP and the reference level for UCH-L1 isbetween at least about 80 pg/mL and about 150 pg/mL, and the referencelevel for GFAP is between about 20 pg/mL and about 200 pg/mL.
 6. Themethod of claim 1, wherein the sample is taken within about 0 to about12 hours after the actual or suspected injury to the head.
 7. A methodcomprising: performing at least one assay for at least one earlybiomarker selected from the group consisting of ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), and ubiquitin carboxy-terminal hydrolase L1 and glial fibrillaryacidic protein in at least a first sample taken at a first time pointand a second sample taken at a second time point obtained from a humansubject after an actual or suspected injury to the head; and a)performing a magnetic resonance imaging (MRI) procedure on the subjectand treating the subject for a moderate, severe, or a moderate to severeTBI when the level of UCH-L1, GFAP, or UCH-L1 and GFAP decreases orincreases from the first sample to the second sample in an amount ofbetween at least about 10 pg/mL and at least about 150 pg/mL and;wherein the sample is whole blood, serum, plasma, or cerebrospinalfluid, and the first time point is within about 24 hours after the headinjury or suspected head injury and the second time point is withinabout 3 to about 6 hours after the first sample is taken.
 8. The methodof claim 7, wherein the assay is selected front the group consisting ofan immunoassay, and a clinical chemistry assay.
 9. The method of claim7, wherein the assay is performed using single molecule detection or apoint-of-care assay.
 10. The method of claim 7, wherein the assay has(a) a sensitivity of at least about 80% and a specificity of at leastabout 30%; or (b) a sensitivity of at least about 75% and a specificityof at least about 40%.
 11. The method of claim 7, wherein (a) the earlybiomarker is UCH-L1 and the level between the first and second sampleincreases or decreases between at least about 30 pg/mL to about 100pg/mL, (b) the at least one early biomarker is GFAP and the levelbetween the first and second sample increases or decreases between atleast about 10 pg/mL to about 150 pg/mL, or (c) the early biomarker isUCH-L1 and GFAP and the level of UCH-L1 between the first and secondsample increases or decreases between at least about 30 pg/mL to about100 pg/mL, and the level of GFAP between the first and second sampleincreases or decreases between at least about 10 pg/mL to about 150pg/mL.
 12. The method of claim 7, wherein the first sample is takenwithin about 0 to about 12 hours after the actual or suspected injury tothe head.
 13. The method of claim 7, wherein the second sample is takenfrom the subject between about 3 hours to about 6 after the firstsample.
 14. The method of claim 7, wherein the level between the firstand second sample for GFAP increases or decreases by about 21 pg/mL,about 34 pg/mL or about 62 pg/mL and the level between the first andsecond sample for UCH-L1 increases or decreases by about 46 pg/mL, about61 pg/mL or about 82 pg/mL.